Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists

ABSTRACT

The present invention relates to dual GLP-1/glucagon receptor agonists and their medical use, for example in the treatment of disorders of the metabolic syndrome, including diabetes and obesity, as well as for reduction of excess food intake.

RELATED APPLICATIONS

This application is a 35 U.S.C. §111(a) filing claiming priority under35 U.S.C. §119(a)-(d) to European Patent Application No.EP2014/05501.01, filed Apr. 7, 2014, the content of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to dual GLP-1/glucagon receptor agonistsand their medical use, for example in the treatment of disorders of themetabolic syndrome, including diabetes and obesity, as well as forreduction of excess food intake. These dual GLP-1/glucagon receptoragonists show reduced activity on the GIP receptor to reduce the risk ofhypoglycemia and are structurally derived from exendin-4, a pure GLP-1receptor agonist.

BACKGROUND OF THE INVENTION

Pocai et al (Obesity 2012; 20:1566-1571; Diabetes 2009, 58, 2258) andDay et al. (Nat Chem Biol 2009; 5:749) describe dual agonists of theglucagon-like peptide-1 (GLP-1) and glucagon receptors, e.g. bycombining the actions of GLP-1 and glucagon in one molecule, which leadto a therapeutic principle with anti-diabetic action and a pronouncedweight lowering effect superior to pure GLP-1 agonists, among others dueto glucagon-receptor mediated increased satiety and energy expenditure.

Holst (Physiol. Rev. 2007, 87, 1409) and Meier (Nat. Rev. Endocrinol.2012, 8, 728) describe that GLP-1 receptor agonists, such as GLP-1,liraglutide and exendin-4, have 3 major pharmacological activities toimprove glycemic control in patients with T2DM by reducing fasting andpostprandial glucose (FPG and PPG): (i) increased glucose-dependentinsulin secretion (improved first- and second-phase), (ii) glucagonsuppressing activity under hyperglycemic conditions, (iii) delay ofgastric emptying rate resulting in retarded absorption of meal-derivedglucose.

The amino acid sequence of GLP-1(7-36)-amide is shown as SEQ ID NO: 2.

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂

Liraglutide is a marketed chemically modified GLP-1 analog in which,among other modifications, a fatty acid is linked to a lysine inposition 20 leading to a prolonged duration of action (Drucker D J etal, Nature Drug Disc. Rev. 9, 267-268, 2010; Buse, J. B. et al., Lancet,374:39-47, 2009).

The amino acid sequence of Liraglutide is shown as SEQ ID NO: 4.

HAEGTFTSDVSSYLEGQAAK((S)-4-Carboxy-4-hexadecanoylamino-butyryl-)EFIAWLVRGRG-OH

Glucagon is a 29-amino acid peptide which is released into thebloodstream when circulating glucose is low. Glucagon's amino acidsequence is shown as SEQ ID NO: 3.

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH

During hypoglycemia, when blood glucose levels drop below normal,glucagon signals the liver to break down glycogen and release glucose,causing an increase of blood glucose levels to reach a normal level.Recent publications suggest that glucagon has in addition beneficialeffects on reduction of body fat mass, reduction of food intake, andincrease of energy expenditure (K M Heppner, Physiology & Behavior 2010,100, 545-548).

GIP (glucose-dependent insulinotropic polypeptide) is a 42 amino acidpeptide that is released from intestinal K-cells following food intake.GIP and GLP-1 are the two gut enteroendocrine cell-derived hormonesaccounting for the incretin effect, which accounts for over 70% of theinsulin response to an oral glucose challenge (Baggio L L, Drucker D J.Biology of incretins: GLP-1 and GIP. Gastroenterology 2007; 132:2131-2157).

GIP's amino acid sequence is shown as SEQ ID NO: 5.

YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-OH

Peptides which are based on the structures of GLP-1 or glucagon, andbind and activate both the glucagon and the GLP-1 receptor (Hjort et al.Journal of Biological Chemistry, 269, 30121-30124, 1994; Day J W et al,Nature Chem Biol, 5: 749-757, 2009) and suppress body weight gain andreduce food intake are described in patent applications WO 2008/071972,WO 2008/101017, WO 2009/155258, WO 2010/096052, WO 2010/096142, WO2011/075393, WO 2008/152403, WO 2010/070251, WO 2010/070252, WO2010/070253, WO 2010/070255, WO 2011/160630, WO 2011/006497, WO2011/087671, WO 2011/087672, WO 2011/117415, WO 2011/117416, WO2012/177443 WO 2012/177444, WO 2012/150503, WO 2013/004983, WO2013/092703, WO 2014/041195 and WO 2014/041375, the contents of whichare herein incorporated by reference. The body weight reduction wasshown to be superior to pure GLP-1 agonists.

In addition, triple co-agonist peptides which not only activate theGLP-1 and the glucagon receptor, but also the GIP receptor are describedin WO 2012/088116 and by V A Gault et al (Biochem Pharmacol, 85,16655-16662, 2013; Diabetologia, 56, 1417-1424, 2013).

Exendin-4 is a 39 amino acid peptide which is produced by the salivaryglands of the Gila monster (Heloderma suspectum) (Eng, J. et al., J.Biol. Chem., 267:7402-05, 1992). Exendin-4 is an activator of the GLP-1receptor, whereas it shows low activation of the GIP receptor and doesnot activate the glucagon receptor (see Table 1).

TABLE 1 Potencies of exendin-4 at human GLP-1, GIP and Glucagonreceptors (indicated in pM) at increasing concentrations and measuringthe formed cAMP as described in Methods. SEQ ID EC50 hGLP-1 R EC50 hGIPR EC50 hGlucagon NO: peptide [PM] [PM] R [pM] 1 exendin-4 0.4 12500.0>10000000

The amino acid sequence of exendin-4 is shown as SEQ ID NO: 1.

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂

Exendin-4 shares many of the glucoregulatory actions observed withGLP-1. Clinical and nonclinical studies have shown that exendin-4 hasseveral beneficial antidiabetic properties including a glucose dependentenhancement in insulin synthesis and secretion, glucose dependentsuppression of glucagon secretion, slowing down gastric emptying,reduction of food intake and body weight, and an increase in beta-cellmass and markers of beta cell function (Gentilella R et al., DiabetesObes Metab., 11:544-56, 2009; Norris S L et al, Diabet Med., 26:837-46,2009; Bunck M C et al, Diabetes Care., 34:2041-7, 2011).

These effects are beneficial not only for diabetics but also forpatients suffering from obesity. Patients with obesity have a higherrisk of getting diabetes, hypertension, hyperlipidemia, cardiovascularand musculoskeletal diseases.

Relative to GLP-1, exendin-4 is resistant to cleavage by dipeptidylpeptidase-4 (DPP4) resulting in a longer half-life and duration ofaction in vivo (Eng J., Diabetes, 45 (Suppl 2):152A (abstract 554),1996).

Exendin-4 was also shown to be much more stable towards degradation byneutral endopeptidase (NEP), when compared to GLP-1, glucagon oroxyntomodulin (Druce M R et al., Endocrinology, 150(4), 1712-1721,2009). Nevertheless, exendin-4 is chemically labile due to methionineoxidation in position 14 (Hargrove D M et al., Regul. Pept., 141: 113-9,2007) as well as deamidation and isomerization of asparagine in position28 (WO 2004/035623).

Compounds of this invention are exendin-4 derivatives, which in additionto the agonistic activity at the GLP-1 receptor of native exendin-4 showagonistic activity at the glucagon receptor and which have—amongothers—the following modification: at position 14 an amino acid carryingan —NH₂ group in the side-chain, which is further substituted with alipophilic residue (e.g. a fatty acid combined with a linker) and atposition 27 an Aib.

Bloom et al. (WO 2006/134340) disclose that peptides which bind andactivate both the glucagon and the GLP-1 receptor can be constructed ashybrid molecules from glucagon and exendin-4, where the N-terminal part(e.g. residues 1-14 or 1-24) originates from glucagon and the C-terminalpart (e.g. residues 15-39 or 25-39) originates from exendin-4. Suchpeptides comprise glucagon's amino acid motif YSKY in position 10-13.Krstenansky et al (Biochemistry, 25, 3833-3839, 1986) show theimportance of these residues 10-13 of glucagon for its receptorinteractions and activation of adenylate cyclase.

In the exendin-4 derivatives described in this invention, several of theunderlying residues are different from glucagon and the peptidesdescribed in WO 2006/134340. In particular residues Tyr10 and Tyr13,which are known to contribute to the fibrillation of glucagon (D EOtzen, Biochemistry, 45, 14503-14512, 2006) are replaced by Leu inposition 10 and Gln, a non-aromatic polar amino acid, in position 13.This replacement, especially in combination with isoleucine in position23 and glutamate in position 24, leads to exendin-4 derivatives withpotentially improved biophysical properties as solubility or aggregationbehaviour in solution. The non-conservative replacement of an aromaticamino acid with a polar amino acid in position 13 of an exendin-4analogue surprisingly leads to peptides with high activity on theglucagon receptor, keeping their activity on the GLP-1 receptor (seealso WO2013/186240.

Furthermore, we surprisingly found that compounds carrying an Aib aminoacid in position 27 show reduced activity on the GIP receptor comparedto the corresponding derivatives with Lys at position 27 as in nativeexendin-4, as shown in Example 5, Table 8. A reduced activation of theGIP receptor is potentially beneficial as there are reports in theliterature that high levels of GIP in diabetics might in some cases leadto more frequent episodes of hypoglycemia (T McLaughlin et al., J ClinEndocrinol Metab, 95, 1851-1855, 2010; A Hadji-Georgopoulos, J ClinEndocrinol Metab, 56, 648-652, 1983).

Furthermore, compounds of this invention are exendin-4 derivatives withfatty acid acylated residues in position 14. This fatty acidfunctionalization in position 14 resulted in exendin-4 derivatives withhigh activity not only at the GLP-1 receptor, but also at the glucagonreceptor, when compared to the corresponding non-acylated exendin-4derivatives, for example those shown in Example 5, Table 7. In addition,this modification results in an improved pharmacokinetic profile.

It is described in the literature (Murage E N et al., Bioorg. Med. Chem.16 (2008), 10106-10112), that a GLP-1 analogue with an acetylated lysineat position 14 showed significantly reduced potency on the GLP-1receptor compared to natural GLP-1.

Compounds of this invention are more resistant to cleavage by neutralendopeptidase (NEP) and dipeptidyl peptidase-4 (DPP4), resulting in alonger half-life and duration of action in vivo, when compared withnative GLP-1 and glucagon.

Compounds of this invention preferably are soluble not only at neutralpH, but also at pH 4.5. This property potentially allows co-formulationfor a combination therapy with an insulin or insulin derivative andpreferably with a basal insulin like insulin glargine/Lantus®.

BRIEF SUMMARY OF THE INVENTION

Native exendin-4 is a pure GLP-1 receptor agonist without activity onthe glucagon receptor and low activity on the GIP receptor. Providedherein are exendin-4 derivatives based on the structure of nativeexendin-4 but differing at ten or more positions as compared to SEQ IDNO: 1 wherein the differences contribute to the enhancement of theagonistic activity at the glucagon receptor. Among othersubstitutions—methionine at position 14 is replaced by an amino acidcarrying an —NH₂ group in the side-chain, which is further substitutedby a lipophilic residue (e.g. a fatty acid combined with a linker).Furthermore, we surprisingly found that a replacement of the lysine atposition 27 by Aib leads to reduced GIP receptor activity compared tothe GLP-1 receptor activity. A reduced activation of the GIP receptor ispotentially beneficial as there are reports in the literature that highlevels of GIP in diabetics might in some cases lead to more frequentepisodes of hypoglycemia (T McLaughlin et al., J Clin Endocrinol Metab,95, 1851-1855, 2010; A Hadji-Georgopoulos, J Clin Endocrinol Metab, 56,648-652, 1983).

The invention provides a peptidic compound having the formula (I):H₂N-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-Asp-Glu-Gln-Arg-Ala-Lys-Leu-Phe-Ile-Glu-Trp-Leu-Aib-X28-X29-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R¹  (I)

-   -   X14 represents an amino acid residue with a functionalized —NH₂        side chain group, selected from the group consisting of Lys,        Orn, Dab, or Dap, wherein the —NH₂ side chain group is        functionalized by —Z—C(O)—R⁵, wherein        -   Z represents a linker in all stereoisomeric forms and        -   R⁵ is a moiety comprising up to 50 carbon atoms and            heteroatoms selected from N and O,    -   X28 represents an amino acid residue selected from Ala, Lys and        Ser,    -   X29 represents an amino acid residue selected from D-Ala and        Gly,    -   R¹ is NH₂ or OH,        -   or a salt or solvate thereof.

The compounds of the invention are GLP-1 and glucagon receptor agonistsas determined by the observation that they are capable of stimulatingintracellular cAMP formation in the assay system described in Methods.

According to another embodiment the compounds of the invention,particularly with a lysine at position 14 which is further substitutedwith a lipophilic residue, exhibit at least a relative activity of 0.1%(i.e. EC₅₀<700 pM), more preferably of 1% (i.e. EC₅₀<70 pM), morepreferably of 5% (i.e. EC₅₀<14 pM) and even more preferably of 10% (i.e.EC₅₀<7 pM) compared to that of GLP-1(7-36)amide at the GLP-1 receptor.Furthermore, the compounds exhibit at least a relative activity of 0.09%(i.e. EC₅₀<1111 pM), more preferably of 0.45% (i.e. EC₅₀<222 pM) andeven more preferably of 0.9% (i.e. EC₅₀<111 pM) compared to that ofnatural glucagon at the glucagon receptor.

The term “activity” as used herein preferably refers to the capabilityof a compound to activate the human GLP-1 receptor and the humanglucagon receptor. More preferably the term “activity” as used hereinrefers to the capability of a compound to stimulate intracellular cAMPformation. The term “relative activity” as used herein is understood torefer to the capability of a compound to activate a receptor in acertain ratio as compared to another receptor agonist or as compared toanother receptor. The activation of the receptors by the agonists (e.g.by measuring the cAMP level) is determined as described herein, e.g. asdescribed in the Example 4.

The compounds of the invention preferably have an EC₅₀ for hGLP-1receptor of 450 pmol or less, preferably of 200 pmol or less, morepreferably of 100 pmol or less, more preferably of 50 pmol or less, morepreferably of 25 pmol or less, more preferably of 10 pmol or less, morepreferably of 8 pmol or less, and more preferably of 5 pmol or lessand/or an EC₅₀ for hGlucagon receptor of 500 pmol or less, preferably of300 pmol or less, more preferably of 200 pmol or less, more preferablyof 150 pmol or less and/or an EC₅₀ for hGIP receptor of 750 pmol ormore, preferably of 1500 pmol or more; more preferably of 2000 pmol ormore. It is particularly preferred that the EC₅₀ for both hGLP-1 andhGlucagon receptors is 250 pm or less, more preferably of 200 pmol orless, more preferably of 150 pmol or less. The EC₅₀ for the hGLP-1receptor, the hGlucagon receptor and the hGIP receptor may be determinedas described in the Methods herein and as used to generate the resultsdescribed in Example 4, Table 6.

The compounds of the invention have the ability to reduce the intestinalpassage, increase the gastric content and/or to reduce the food intakeof a patient. These activities of the compounds of the invention can beassessed in animal models known to the skilled person and also describedherein in the Methods.

The compounds of the invention have the ability to reduce blood glucoselevel, and/or to reduce HbA1c levels of a patient. These activities ofthe compounds of the invention can be assessed in animal models known tothe skilled person and also described herein in the Methods.

The compounds of the invention have the ability to reduce body weight ofa patient. These activities of the compounds of the invention can beassessed in animal models known to the skilled person and also describedherein in the Methods and in Example 7.

It was found that peptidic compounds of the formula (I) particularlythose with a lysine at position 14 which is further substituted with alipophilic residue, showed increased glucagon receptor activationcompared to derivatives having the original methionine (from exendin-4)or leucine at position 14 (see Table 7). Furthermore, oxidation (invitro or in vivo) of methionine is not possible anymore.

It was also found that compounds carrying an Aib amino acid in position27 show reduced activity on the GIP receptor compared to thecorresponding derivatives with Lys at position 27 as in nativeexendin-4, as shown in Example 5, Table 8. A reduced activation of theGIP receptor is potentially beneficial as there are reports in theliterature that high levels of GIP in diabetics might in some cases leadto more frequent episodes of hypoglycemia (T McLaughlin et al., J ClinEndocrinol Metab, 95, 1851-1855, 2010; A Hadji-Georgopoulos, J ClinEndocrinol Metab, 56, 648-652, 1983).

In one embodiment the compounds of the invention have a high solubilityat acidic and/or physiological pH values, e.g., at pH 4.5 and/or at pH7.4 at 25° C., in another embodiment at least 1 mg/ml and in aparticular embodiment at least 5 mg/ml.

Furthermore, the compounds of the invention preferably have a highstability when stored in solution. Preferred assay conditions fordetermining the stability is storage for 7 days at 40° C. in solution atpH 4.5 or pH 7.4. The remaining amount of peptide is determined bychromatographic analyses as described in the Examples. Preferably, after7 days at 40° C. in solution at pH 4.5 or pH 7.4 the remaining peptideis at least 75%, more preferably at least 80%, even more preferably atleast 85% and even more preferably at least 90%.

Preferably, the compounds of the present invention comprise a peptidemoiety which is a linear sequence of 39 amino carboxylic acids,particularly α-amino carboxylic acids linked by peptide, i.e.carboxamide bonds.

In one embodiment, R¹ is NH₂ and in a further embodiment R¹ is OH.

Specific preferred examples for —Z—C(O)—R⁵ groups are listed in thefollowing Table 2, which are selected from

(S)-4-Carboxy-4-hexadecanoylamino-butyryl-,(S)-4-Carboxy-4-octadecanoylamino-butyryl-,(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,[2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-,(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-(17-carboxy-heptadecanoyl)amino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl.

Further preferred are stereoisomers, particularly enantiomers of thesegroups, either S- or R-enantiomers. The term “R” in Table 2 is intendedto mean the attachment site of —Z—C(O)—R⁵ at the peptide back bone, i.e.particularly the ε-amino group of Lys.

TABLE 2 Structure/IUPAC name

E-x70

E-x53

E-E-x53

AEEAc- AEEAc- E-x53

AEEAc- AEEAc- E-x70

AEEAc- AEEAc- AEEAc- x70

AEEAc- AEEAc- E-x99

A further embodiment relates to a group of compounds, wherein

X14 represents Lys wherein the —NH₂ side chain group is functionalizedwith a group —Z—C(O)R⁵, wherein

Z represents a group selected from γE, γE-γE, AEEAc-AEEAc-γE andAEEAc-AEEAc-AEEAc and

R⁵ represents a group selected from pentadecanyl, heptadecanyl or16-carboxy-hexadecanyl.

A further embodiment relates to a group of compounds, wherein

X14 represents Lys wherein the —NH₂ side chain group is functionalizedwith a group —Z—C(O)R⁵, wherein

Z represents a group selected from γE, γE-γE, AEEAc-AEEAc-γE andAEEAc-AEEAc-AEEAc and

R⁵ represents a group selected from pentadecanyl or heptadecanyl.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by (S)-4-Carboxy-4-octadecanoylamino-butyryl-,    -   X28 represents Ala,    -   X29 represents an amino acid residue selected from Gly and        D-Ala,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,    -   X28 represents Ala,    -   X29 represents an amino acid residue selected from Gly and        D-Ala,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,    -   X28 represents an amino acid residue selected from Ala, Ser and        Lys,    -   X29 represents an amino acid residue selected from Gly and        D-Ala,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,        [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-(17-carboxy-heptadecanoyl)amino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,    -   X28 represents Ala,    -   X29 represents an amino acid residue selected from D-Ala and        Gly,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,        [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-,    -   X28 represents Ala,    -   X29 represents an amino acid residue selected from D-Ala and        Gly,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,    -   X28 represents Ser,    -   X29 represents an amino acid residue selected from D-Ala and        Gly,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,    -   X28 represents Lys,    -   X29 represents an amino acid residue selected from D-Ala and        Gly,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,    -   X28 represents an amino acid residue selected from Ala, Lys and        Ser,    -   X29 represents D-Ala,    -   R¹ represents NH₂,    -   or a salt or solvate thereof.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,        [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-(17-carboxy-heptadecanoyl)amino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,    -   X28 represents an amino acid residue selected from Ala, Lys and        Ser,    -   X29 represents Gly,    -   R¹ represents NH₂.    -   or a salt or solvate thereof.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,        (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,        [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-,    -   X28 represents an amino acid residue selected from Ala, Lys and        Ser,    -   X29 represents Gly,    -   R¹ represents NH₂.    -   or a salt or solvate thereof.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,    -   X28 represents Ala,    -   X29 represents an amino acid residue selected from Gly and        D-Ala,    -   R¹ represents NH₂.    -   or a salt or solvate thereof.

A further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys, wherein the —NH₂ side chain group is        functionalized by (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,        (S)-4-Carboxy-4-octadecanoylamino-butyryl-,        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-    -   or a salt or solvate thereof.

A still further embodiment relates to a group of compounds, wherein

-   -   X14 represents Lys, wherein the —NH₂ side chain group is        functionalized by        (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-.    -   or a salt or solvate thereof.

Specific examples of peptidic compounds of formula (I) are the compoundsof SEQ ID NO: 6-19, as well as salts or solvates thereof.

Specific examples of peptidic compounds of formula (I) are the compoundsof SEQ ID NO: 6-18, as well as salts or solvates thereof.

Specific examples of peptidic compounds of formula (I) are the compoundsof SEQ ID NO: 7 and 8 as well as salts or solvates thereof.

In certain embodiments, i.e. when the compound of formula (I) comprisesgenetically encoded amino acid residues, the invention further providesa nucleic acid (which may be DNA or RNA) encoding said compound, anexpression vector comprising such a nucleic acid, and a host cellcontaining such a nucleic acid or expression vector.

In a further aspect, the present invention provides a compositioncomprising a compound of the invention in admixture with a carrier. Inpreferred embodiments, the composition is a pharmaceutically acceptablecomposition and the carrier is a pharmaceutically acceptable carrier.The compound of the invention may be in the form of a salt, e.g. apharmaceutically acceptable salt or a solvate, e.g. a hydrate. In stilla further aspect, the present invention provides a composition for usein a method of medical treatment, particularly in human medicine.

In certain embodiments, the nucleic acid or the expression vector may beused as therapeutic agents, e.g. in gene therapy.

The compounds of formula (I) are suitable for therapeutic applicationwithout an additional therapeutically effective agent. In otherembodiments, however, the compounds are used together with at least oneadditional therapeutically active agent, as described in “combinationtherapy”.

The compounds of formula (I) are particularly suitable for the treatmentor prevention of diseases or disorders caused by, associated with and/oraccompanied by disturbances in carbohydrate and/or lipid metabolism,e.g. for the treatment or prevention of hyperglycemia, type 2 diabetes,impaired glucose tolerance, type 1 diabetes, obesity and metabolicsyndrome. Further, the compounds of the invention are particularlysuitable for the treatment or prevention of degenerative diseases,particularly neurodegenerative diseases.

The compounds described find use, inter alia, in preventing weight gainor promoting weight loss. By “preventing” is meant inhibiting orreducing when compared to the absence of treatment, and is notnecessarily meant to imply complete cessation of a disorder.

The compounds of the invention may cause a decrease in food intakeand/or increase in energy expenditure, resulting in the observed effecton body weight.

Independently of their effect on body weight, the compounds of theinvention may have a beneficial effect on circulating cholesterollevels, being capable of improving lipid levels, particularly LDL, aswell as HDL levels (e.g. increasing HDL/LDL ratio).

Thus, the compounds of the invention can be used for direct or indirecttherapy of any condition caused or characterised by excess body weight,such as the treatment and/or prevention of obesity, morbid obesity,obesity linked inflammation, obesity linked gallbladder disease, obesityinduced sleep apnea. They may also be used for treatment and preventionof the metabolic syndrome, diabetes, hypertension, atherogenicdyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease,or stroke. Their effects in these conditions may be as a result of orassociated with their effect on body weight, or may be independentthereof.

Preferred medical uses include delaying or preventing diseaseprogression in type 2 diabetes, treating metabolic syndrome, treatingobesity or preventing overweight, for decreasing food intake, increaseenergy expenditure, reducing body weight, delaying the progression fromimpaired glucose tolerance (IGT) to type 2 diabetes; delaying theprogression from type 2 diabetes to insulin-requiring diabetes;regulating appetite; inducing satiety; preventing weight regain aftersuccessful weight loss; treating a disease or state related tooverweight or obesity; treating bulimia; treating binge eating; treatingatherosclerosis, hypertension, type 2 diabetes, IGT, dyslipidemia,coronary heart disease, hepatic steatosis, treatment of beta-blockerpoisoning, use for inhibition of the motility of the gastrointestinaltract, useful in connection with investigations of the gastrointestinaltract using techniques such as X-ray, CT- and NMR-scanning.

Further preferred medical uses include treatment or prevention ofdegenerative disorders, particularly neurodegenerative disorders such asAlzheimer's disease, Parkinson's disease, Huntington's disease, ataxia,e.g spinocerebellar ataxia, Kennedy disease, myotonic dystrophy, Lewybody dementia, multi-systemic atrophy, amyotrophic lateral sclerosis,primary lateral sclerosis, spinal muscular atrophy, prion-associateddiseases, e.g. Creutzfeldt-Jacob disease, multiple sclerosis,telangiectasia, Batten disease, corticobasal degeneration, subacutecombined degeneration of spinal cord, Tabes dorsalis, Tay-Sachs disease,toxic encephalopathy, infantile Refsum disease, Refsum disease,neuroacanthocytosis, Niemann-Pick disease, Lyme disease, Machado-Josephdisease, Sandhoff disease, Shy-Drager syndrome, wobbly hedgehogsyndrome, proteopathy, cerebral β-amyloid angiopathy, retinal ganglioncell degeneration in glaucoma, synucleinopathies, tauopathies,frontotemporal lobar degeneration (FTLD), dementia, cadasil syndrome,hereditary cerebral hemorrhage with amyloidosis, Alexander disease,seipinopathies, familial amyloidotic neuropathy, senile systemicamyloidosis, serpinopathies, AL (light chain) amyloidosis (primarysystemic amyloidosis), AH (heavy chain) amyloidosis, AA (secondary)amyloidosis, aortic medial amyloidosis, ApoAI amyloidosis, ApoAIIamyloidosis, ApoAIV amyloidosis, familial amyloidosis of the Finnishtype (FAF), Lysozyme amyloidosis, Fibrinogen amyloidosis, Dialysisamyloidosis, Inclusion body myositis/myopathy, Cataracts, Retinitispigmentosa with rhodopsin mutations, medullary thyroid carcinoma,cardiac atrial amyloidosis, pituitary prolactinoma, Hereditary latticecorneal dystrophy, Cutaneous lichen amyloidosis, Mallory bodies, corneallactoferrin amyloidosis, pulmonary alveolar proteinosis, odontogenic(Pindborg) tumor amyloid, cystic fibrosis, sickle cell disease orcritical illness myopathy (CIM).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The amino acid sequences of the present invention contain theconventional one letter and three letter codes for naturally occuringamino acids, as well as generally accepted three letter codes for otheramino acids, such as Aib (α-aminoisobutyric acid).

The term “native exendin-4” refers to native exendin-4 having thesequence

(SEQ ID NO: 1) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂. 

The invention provides peptidic compounds as defined above.

The peptidic compounds of the present invention comprise a linearbackbone of amino carboxylic acids linked by peptide, i.e. carboxamidebonds. Preferably, the amino carboxylic acids are α-amino carboxylicacids and more preferably L-α-amino carboxylic acids, unless indicatedotherwise. The peptidic compounds preferably comprise a backbonesequence of 39 amino carboxylic acids.

The peptidic compounds of the present invention may have unmodifiedside-chains, but carry at least one modification at one of the sidechains.

For the avoidance of doubt, in the definitions provided herein, it isgenerally intended that the sequence of the peptidic moiety (I) differsfrom native exendin-4 at least at one of those positions which arestated to allow variation. Amino acids within the peptide moiety (I) canbe considered to be numbered consecutively from 1 to 39 in theconventional N-terminal to C-terminal direction. Reference to a“position” within peptidic moiety (I) should be constructed accordingly,as should reference to positions within native exendin-4 and othermolecules, e.g., in exendin-4, His is at position 1, Gly at position 2,. . . , Met at position 14, . . . and Ser at position 39.

An amino acid residue with an —NH₂ side chain group, e.g. Lys, Orn, Dabor Dap, is functionalized in that at least one H atom of the —NH₂ sidechain group is replaced by —Z—C(O)—R⁵, wherein R⁵ comprises a lipophilicmoiety, e.g. an acyclic linear or branched (C₈-C₃₀) saturated orunsaturated hydrocarbon group, which is unsubstituted or substitutede.g. by halogen, —OH and/or CO₂H and Z comprises a linker in allstereoisomeric forms, e.g. a linker comprising one or more, e.g. 1 to 5,preferably 1, 2 or 3 amino acid linker groups selected from the groupγ-Glutamate (γE) and AEEAc. Preferred groups R⁵ comprise a lipophilicmoiety, e.g. an acyclic linear or branched (C₁₂-C₂₀) saturated orunsaturated hydrocarbon group, e.g. pentadecanyl, hexadecanyl orheptadecanyl, which is unsubstituted or substituted by CO₂H, morepreferably pentadecanyl, heptadecanyl or 16-carboxy-hexadecanyl. In oneembodiment amino acid linker groups are selected from γE, γE-γE,AEEAc-AEEAc-γE and AEEAc-AEEAc-AEEAc. In another embodiment the aminoacid linker group is γE. In another embodiment the amino acid linkergroup is γE-γE. In another embodiment the amino acid linker group isAEEAc-AEEAc-γE. In another embodiment the amino acid linker group isAEEAc-AEEAc-AEEAc.

In a further aspect, the present invention provides a compositioncomprising a compound of the invention as described herein, or a salt orsolvate thereof, in admixture with a carrier.

The invention also provides the use of a compound of the presentinvention for use as a medicament, particularly for the treatment of acondition as described below.

The invention also provides a composition wherein the composition is apharmaceutically acceptable composition, and the carrier is apharmaceutically acceptable carrier.

Peptide Synthesis

The skilled person is aware of a variety of different methods to preparepeptides that are described in this invention. These methods include butare not limited to synthetic approaches and recombinant gene expression.Thus, one way of preparing these peptides is the synthesis in solutionor on a solid support and subsequent isolation and purification. Adifferent way of preparing the peptides is gene expression in a hostcell in which a DNA sequence encoding the peptide has been introduced.Alternatively, the gene expression can be achieved without utilizing acell system. The methods described above may also be combined in anyway.

A preferred way to prepare the peptides of the present invention issolid phase synthesis on a suitable resin. Solid phase peptide synthesisis a well-established methodology (see for example: Stewart and Young,Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill.,1984; E. Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis. APractical Approach, Oxford-IRL Press, New York, 1989). Solid phasesynthesis is initiated by attaching an N-terminally protected amino acidwith its carboxy terminus to an inert solid support carrying a cleavablelinker. This solid support can be any polymer that allows coupling ofthe initial amino acid, e.g. a trityl resin, a chlorotrityl resin, aWang resin or a Rink resin in which the linkage of the carboxy group (orcarboxamide for Rink resin) to the resin is sensitive to acid (when Fmocstrategy is used). The polymer support must be stable under theconditions used to deprotect the α-amino group during the peptidesynthesis.

After the first amino acid has been coupled to the solid support, theα-amino protecting group of this amino acid is removed. The remainingprotected amino acids are then coupled one after the other in the orderrepresented by the peptide sequence using appropriate amide couplingreagents, for example BOP, HBTU, HATU or DIC(N,N′-diisopropylcarbodiimide)/HOBt (1-hydroxybenzotriazole), whereinBOP, HBTU and HATU are used with tertiary amine bases. Alternatively,the liberated N-terminus can be functionalized with groups other thanamino acids, for example carboxylic acids, etc.

Usually, reactive side-chain groups of the amino acids are protectedwith suitable blocking groups. These protecting groups are removed afterthe desired peptides have been assembled. They are removed concomitantlywith the cleavage of the desired product from the resin under the sameconditions. Protecting groups and the procedures to introduce protectinggroups can be found in Protective Groups in Organic Synthesis, 3d ed.,Greene, T. W. and Wuts, P. G. M., Wiley & Sons (New York: 1999).

In some cases it might be desirable to have side-chain protecting groupsthat can selectively be removed while other side-chain protecting groupsremain intact. In this case the liberated functionality can beselectively functionalized. For example, a lysine may be protected withan ivDde ([1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl)protecting group (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998),1603) which is labile to a very nucleophilic base, for example 4%hydrazine in DMF (dimethyl formamide). Thus, if the N-terminal aminogroup and all side-chain functionalities are protected with acid labileprotecting groups, the ivDde group can be selectively removed using 4%hydrazine in DMF and the corresponding free amino group can then befurther modified, e.g. by acylation. The lysine can alternatively becoupled to a protected amino acid and the amino group of this amino acidcan then be deprotected resulting in another free amino group which canbe acylated or attached to further amino acids.

Finally the peptide is cleaved from the resin. This can be achieved byusing King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J.Peptide Protein Res. 36, 1990, 255-266). The raw material can then bepurified by chromatography, e.g. preparative RP-HPLC, if necessary.

Potency

As used herein, the term “potency” or “in vitro potency” is a measurefor the ability of a compound to activate the receptors for GLP-1,glucagon or GIP in a cell-based assay. Numerically, it is expressed asthe “EC50 value”, which is the effective concentration of a compoundthat induces a half maximal increase of response (e.g. formation ofintracellular cAMP) in a dose-response experiment.

Therapeutic Uses

Metabolic syndrome is a combination of medical disorders that, whenoccurring together, increase the risk of developing type 2 diabetes, aswell as atherosclerotic vascular disease, e.g. heart disease and stroke.Defining medical parameters for the metabolic syndrome include diabetesmellitus, impaired glucose tolerance, raised fasting glucose, insulinresistance, urinary albumin secretion, central obesity, hypertension,elevated triglycerides, elevated LDL cholesterol and reduced HDLcholesterol.

Obesity is a medical condition in which excess body fat has accumulatedto the extent that it may have an adverse effect on health and lifeexpectancy and due to its increasing prevalence in adults and childrenit has become one of the leading preventable causes of death in modernworld. It increases the likelihood of various other diseases, includingheart disease, type 2 diabetes, obstructive sleep apnoe, certain typesof cancer, as well as osteoarthritis, and it is most commonly caused bya combination of excess food intake, reduced energy expenditure, as wellas genetic susceptibility.

Diabetes mellitus, often simply called diabetes, is a group of metabolicdiseases in which a person has high blood sugar levels, either becausethe body does not produce enough insulin, or because cells do notrespond to the insulin that is produced. The most common types ofdiabetes are: (1) type 1 diabetes, where the body fails to produceinsulin; (2) type 2 diabetes (T2DM), where the body fails to use insulinproperly, combined with an increase in insulin deficiency over time, and(3) gestational diabetes, where women develop diabetes due to theirpregnancy. All forms of diabetes increase the risk of long-termcomplications, which typically develop after many years. Most of theselong-term complications are based on damage to blood vessels and can bedivided into the two categories “macrovascular” disease, arising fromatherosclerosis of larger blood vessels and “microvascular” disease,arising from damage of small blood vessels. Examples for macrovasculardisease conditions are ischemic heart disease, myocardial infarction,stroke and peripheral vascular disease. Examples for microvasculardiseases are diabetic retinopathy, diabetic nephropathy, as well asdiabetic neuropathy.

The receptors for GLP-1 and GIP as well as glucagon are members of thefamily of 7-transmembrane-spanning, heterotrimeric G-protein coupledreceptors. They are structurally related to each other and share notonly a significant level of sequence identity, but have also similarmechanisms of ligand recognition and intracellular signaling pathways.

Similarly, the peptides GLP-1, GIP and glucagon share regions of highsequence identity/similarity. GLP-1 and glucagon are produced from acommon precursor, preproglucagon, which is differentially processed in atissue-specific manner to yield e.g. GLP-1 in intestinal endocrine cellsand glucagon in alpha cells of pancreatic islets. GIP is derived from alarger proGIP prohormone precursor and is synthesized and released fromK-cells located in the small intestine.

The peptidic incretin hormones GLP-1 and GIP are secreted by intestinalendocrine cells in response to food and account for up to 70% ofmeal-stimulated insulin secretion. Evidence suggests that GLP-1secretion is reduced in subjects with impaired glucose tolerance or type2 diabetes, whereas responsiveness to GLP-1 is still preserved in thesepatients. Thus, targeting of the GLP-1 receptor with suitable agonistsoffers an attractive approach for treatment of metabolic disorders,including diabetes. The receptor for GLP-1 is distributed widely, beingfound mainly in pancreatic islets, brain, heart, kidney and thegastrointestinal tract. In the pancreas, GLP-1 acts in a strictlyglucose-dependent manner by increasing secretion of insulin from betacells. This glucose-dependency shows that activation of GLP-1 receptorsis unlikely to cause hypoglycemia. Also the receptor for GIP is broadlyexpressed in peripheral tissues including pancreatic islets, adiposetissue, stomach, small intestine, heart, bone, lung, kidney, testis,adrenal cortex, pituitary, endothelial cells, trachea, spleen, thymus,thyroid and brain. Consistent with its biological function as incretinhormone, the pancreatic β-cell express the highest levels of thereceptor for GIP in humans. There is some clinical evidence that theGIP-receptor mediated signaling could be impaired in patients with T2DMbut GIP-action is shown to be reversible and can be restored withimprovement of the diabetic status. While there are many reports thatalso GIP action on insulin secretion is glucose-dependent, there arealso reports in the literature that high plasma levels of GIP might leadto more frequent episodes of hypoglycemia (T McLaughlin et al., J ClinEndocrinol Metab, 95, 1851-1855, 2010; A Hadji-Georgopoulos, J ClinEndocrinol Metab, 56, 648-652, 1983). In addition, plasma GIP levels inobese subjects were reported to be higher than normal, suggesting thatGIP might induce obesity and insulin resistance (W Creutzfeldt et al.Diabetologia. 1978, 14, 15-24). This is supported by reports that theablation of the GIP receptor might prevent those conditions: GIPreceptor knock-out mice fed on high-fat diet actually showed asuppression of body weight compared to wild-type mice (K Miyawaki et al.Nat Med. 2002, 8, 738-42), and long-term administration of the GIPreceptor antagonist (Pro3)GIP also prevented obesity and insulinresistance in mice (V A Gault et al. Diabetologia. 2007, 50, 1752-62).Therefore, goal of this invention was to provide dual GLP-1/glucagonreceptor agonists with reduced activity on the GIP receptor.

Glucagon is a 29 amino acid peptide hormone that is produced bypancreatic alpha cells and released into the bloodstream whencirculating glucose is low. An important physiological role of glucagonis to stimulate glucose output in the liver, which is a processproviding the major counterregulatory mechanism for insulin inmaintaining glucose homeostasis in vivo.

Glucagon receptors are however also expressed in extra-hepatic tissuessuch as kidney, heart, adipocytes, lymphoblasts, brain, retina, adrenalgland and gastrointestinal tract, suggesting a broader physiologicalrole beyond glucose homeostasis. Accordingly, recent studies havereported that glucagon has therapeutically positive effects on energymanagement, including stimulation of energy expenditure andthermogenesis, accompanied by reduction of food intake and body weightloss. Altogether, stimulation of glucagon receptors might be useful inthe treatment of obesity and the metabolic syndrome.

Oxyntomodulin is a peptide hormone consisting of glucagon with an eightamino acids encompassing C-terminal extension. Like GLP-1 and glucagon,it is pre-formed in preproglucagon and cleaved and secreted in atissue-specific manner by endocrinal cells of the small bowel.Oxyntomodulin is known to stimulate both, the receptors for GLP-1 andglucagon and is therefore the prototype of a dual agonist.

As GLP-1 is known for its anti-diabetic effects, GLP-1 and glucagon areboth known for their food intake-suppressing effects and glucagon isalso a mediator of additional energy expenditure, it is conceivable thata combination of the activities of the two hormones in one molecule canyield a powerful medication for treatment of the metabolic syndrome andin particular its components diabetes and obesity.

Accordingly, the compounds of the invention may be used for treatment ofglucose intolerance, insulin resistance, pre-diabetes, increased fastingglucose (hyperglycemia), type 2 diabetes, hypertension, dyslipidemia,arteriosclerosis, coronary heart disease, peripheral artery disease,stroke or any combination of these individual disease components.

In addition, they may be used for control of appetite, feeding andcalory intake, increase of energy expenditure, prevention of weightgain, promotion of weight loss, reduction of excess body weight andaltogether treatment of obesity, including morbid obesity.

The compounds of the invention are agonists for the receptors for GLP-1and for glucagon (e.g. “dual agonists”) with reduced activity on the GIPreceptor and may provide therapeutic benefit to address a clinical needfor targeting the metabolic syndrome by allowing simultaneous treatmentof diabetes and obesity.

Further disease states and health conditions which could be treated withthe compounds of the invention are obesity-linked inflammation,obesity-linked gallbladder disease and obesity-induced sleep apnea.

Although all these conditions could be associated directly or indirectlywith obesity, the effects of the compounds of the invention may bemediated in whole or in part via an effect on body weight, orindependent thereof.

Further, diseases to be treated are neurodegenerative diseases such asAlzheimer's disease or Parkinson's disease, or other degenerativediseases as described above.

In one embodiment the compounds are useful in the treatment orprevention of hyperglycemia, type 2 diabetes, obesity.

Compared to GLP-1, glucagon and oxyntomodulin, exendin-4 has beneficialphysicochemical properties, such as solubility and stability in solutionand under physiological conditions (including enzymatic stabilitytowards degradation by enzymes, such as DPP4 or NEP), which results in alonger duration of action in vivo. Therefore, the pure GLP-1 receptoragonist exendin-4 might serve as good starting scaffold to obtainexendin-4 analogues with dual GLP-1/glucagon receptor agonism.

Nevertheless, also exendin-4 has been shown to be chemically labile dueto methionine oxdiation in position 14 as well as deamidation andisomerization of asparagine in position 28. Therefore, stability mightbe further improved by substitution of methionine at position 14 and theavoidance of sequences that are known to be prone to degradation viaaspartimide formation, especially Asp-Gly or Asn-Gly at positions 28 and29.

Pharmaceutical Compositions

The term “pharmaceutical composition” indicates a mixture containingingredients that are compatible when mixed and which may beadministered. A pharmaceutical composition may include one or moremedicinal drugs. Additionally, the pharmaceutical composition mayinclude carriers, buffers, acidifying agents, alkalizing agents,solvents, adjuvants, tonicity adjusters, emollients, expanders,preservatives, physical and chemical stabilizers e.g. surfactants,antioxidants and other components, whether these are considered activeor inactive ingredients. Guidance for the skilled in preparingpharmaceutical compositions may be found, for example, in Remington: TheScience and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A. R.,2000, Lippencott Williams & Wilkins and in R. C. Rowe et al (Ed),Handbook of Pharmaceutical Excipients, PhP, May 2013 update.

The exendin-4 peptide derivatives of the present invention, or saltsthereof, are administered in conjunction with an acceptablepharmaceutical carrier, diluent, or excipient as part of apharmaceutical composition. A “pharmaceutically acceptable carrier” is acarrier which is physiologically acceptable (e.g. physiologicallyacceptable pH) while retaining the therapeutic properties of thesubstance with which it is administered. Standard acceptablepharmaceutical carriers and their formulations are known to one skilledin the art and described, for example, in Remington: The Science andPractice of Pharmacy, (20th ed.) ed. A. R. Gennaro A. R., 2000,Lippencott Williams & Wilkins and in R. C. Rowe et al (Ed), Handbook ofPharmaceutical excipients, PhP, May 2013 update. One exemplarypharmaceutically acceptable carrier is physiological saline solution.

In one embodiment carriers are selected from the group of buffers (e.g.citrate/citric acid), acidifying agents (e.g. hydrochloric acid),alkalizing agents (e.g. sodium hydroxide), preservatives (e.g. phenol),co-solvents (e.g. polyethylene glycol 400), tonicity adjusters (e.g.mannitol), stabilizers (e.g. surfactant, antioxidants, amino acids).

Concentrations used are in a range that is physiologically acceptable.

Acceptable pharmaceutical carriers or diluents include those used informulations suitable for oral, rectal, nasal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, and transdermal)administration. The compounds of the present invention will typically beadministered parenterally.

The term “pharmaceutically acceptable salt” means salts of the compoundsof the invention which are safe and effective for use in mammals.Pharmaceutically acceptable salts may include, but are not limited to,acid addition salts and basic salts. Examples of acid addition saltsinclude chloride, sulfate, hydrogen sulfate, (hydrogen) phosphate,acetate, citrate, tosylate or mesylate salts. Examples of basic saltsinclude salts with inorganic cations, e.g. alkaline or alkaline earthmetal salts such as sodium, potassium, magnesium or calcium salts andsalts with organic cations such as amine salts. Further examples ofpharmaceutically acceptable salts are described in Remington: TheScience and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A. R.,2000, Lippencott Williams & Wilkins or in Handbook of PharmaceuticalSalts, Properties, Selection and Use, e.d. P. H. Stahl, C. G. Wermuth,2002, jointly published by Verlag Helvetica Chimica Acta, Zurich,Switzerland, and Wiley-VCH, Weinheim, Germany.

The term “solvate” means complexes of the compounds of the invention orsalts thereof with solvent molecules, e.g. organic solvent moleculesand/or water.

In the pharmaceutical composition, the exendin-4 derivative can be inmonomeric or oligomeric form.

The term “therapeutically effective amount” of a compound refers to anontoxic but sufficient amount of the compound to provide the desiredeffect. The amount of a compound of the formula (I) necessary to achievethe desired biological effect depends on a number of factors, forexample the specific compound chosen, the intended use, the mode ofadministration and the clinical condition of the patient. An appropriate“effective” amount in any individual case may be determined by one ofordinary skill in the art using routine experimentation. For example the“therapeutically effective amount” of a compound of the formula (I) isabout 0.01 to 50 mg/dose, preferably 0.1 to 10 mg/dose.

Pharmaceutical compositions of the invention are those suitable forparenteral (for example subcutaneous, intramuscular, intradermal orintravenous), oral, rectal, topical and peroral (for example sublingual)administration, although the most suitable mode of administrationdepends in each individual case on the nature and severity of thecondition to be treated and on the nature of the compound of formula (I)used in each case.

Suitable pharmaceutical compositions may be in the form of separateunits, for example capsules, tablets and powders in vials or ampoules,each of which contains a defined amount of the compound; as powders orgranules; as solution or suspension in an aqueous or nonaqueous liquid;or as an oil-in-water or water-in-oil emulsion. It may be provided insingle or multiple dose injectable form, for example in the form of apen. The compositions may, as already mentioned, be prepared by anysuitable pharmaceutical method which includes a step in which the activeingredient and the carrier (which may consist of one or more additionalingredients) are brought into contact.

In certain embodiments the pharmaceutical composition may be providedtogether with a device for application, for example together with asyringe, an injection pen or an autoinjector. Such devices may beprovided separate from a pharmaceutical composition or prefilled withthe pharmaceutical composition.

Combination Therapy

The compounds of the present invention, dual agonists for the GLP-1 andglucagon receptors, can be widely combined with other pharmacologicallyactive compounds, such as all drugs mentioned in the Rote Liste 2014,e.g. with all weight-reducing agents or appetite suppressants mentionedin the Rote Liste 2014, chapter 1, all lipid-lowering agents mentionedin the Rote Liste 2014, chapter 58, all antihypertensives andnephroprotectives, mentioned in the Rote Liste 2014, or all diureticsmentioned in the Rote Liste 2014, chapter 36.

The active ingredient combinations can be used especially for asynergistic improvement in action. They can be applied either byseparate administration of the active ingredients to the patient or inthe form of combination products in which a plurality of activeingredients are present in one pharmaceutical preparation. When theactive ingredients are administered by separate administration of theactive ingredients, this can be done simultaneously or successively.

Most of the active ingredients mentioned hereinafter are disclosed inthe USP Dictionary of USAN and International Drug Names, USPharmacopeia, Rockville 2011.

Other active substances which are suitable for such combinations includein particular those which for example potentiate the therapeutic effectof one or more active substances with respect to one of the indicationsmentioned and/or which allow the dosage of one or more active substancesto be reduced.

Therapeutic agents which are suitable for combinations include, forexample, antidiabetic agents such as:

Insulin and Insulin derivatives, for example: Glargine/Lantus®, 270-330U/mL of insulin glargine (EP 2387989 A), 300 U/mL of insulin glargine(EP 2387989 A), Glulisin/Apidra®, Detemir/Levemir®,Lispro/Humalog®/Liprolog®, Degludec/DegludecPlus, Aspart, basal insulinand analogues (e.g. LY-2605541, LY2963016, NN1436), PEGylated insulinLispro, Humulin®, Linjeta, SuliXen®, NN1045, Insulin plus Symlin,PE0139, fast-acting and short-acting insulins (e.g. Linjeta, PH20,NN1218, HinsBet), (APC-002)hydrogel, oral, inhalable, transdermal andsublingual insulins (e.g. Exubera®, Nasulin®, Afrezza, Tregopil, TPM 02,Capsulin, Oral-Lyn®, Cobalamin® oral insulin, ORMD-0801, NN1953, NN1954,NN1956, VIAtab, Oshadi oral insulin). Additionally included are alsothose insulin derivatives which are bonded to albumin or another proteinby a bifunctional linker.

GLP-1, GLP-1 analogues and GLP-1 receptor agonists, for example:Lixisenatide/AVE0010/ZP10/Lyxumia,Exenatide/Exendin-4/Byetta/Bydureon/ITCA 650/AC-2993,Liraglutide/Victoza, Semaglutide, Taspoglutide, Syncria/Albiglutide,Dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054,Langlenatide/HM-11260C, CM-3, GLP-1 Eligen, ORMD-0901, NN-9924, NN-9926,NN-9927, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, GSK-2374697,DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022, TT-401, BHM-034. MOD-6030,CAM-2036, DA-15864, ARI-2651, ARI-2255, Exenatide-XTEN andGlucagon-Xten.

DPP4 inhibitors, for example: Alogliptin/Nesina,Trajenta/Linagliptin/BI-1356/Ondero/Trajenta/Tradjenta/Trayenta/Tradzenta,Saxagliptin/Onglyza,Sitagliptin/Januvia/Xelevia/Tesave/Janumet/Velmetia,Galvus/Vildagliptin, Anagliptin, Gemigliptin, Teneligliptin,Melogliptin, Trelagliptin, DA-1229, Omarigliptin/MK-3102, KM-223,Evogliptin, ARI-2243, PBL-1427, Pinoxacin.

SGLT2 inhibitors, for example: Invokana/Canaglifozin,Forxiga/Dapagliflozin, Remoglifozin, Sergliflozin, Empagliflozin,Ipragliflozin, Tofogliflozin, Luseogliflozin, LX-4211,Ertuglifozin/PF-04971729, RO-4998452, EGT-0001442, KGA-3235/DSP-3235,LIK066, SBM-TFC-039, Biguanides (e.g. Metformin, Buformin, Phenformin),Thiazolidinediones (e.g. Pioglitazone, Rivoglitazone, Rosiglitazone,Troglitazone), dual PPAR agonists (e.g. Aleglitazar, Muraglitazar,Tesaglitazar), Sulfonylureas (e.g. Tolbutamide, Glibenclamide,Glimepiride/Amaryl, Glipizide), Meglitinides (e.g. Nateglinide,Repaglinide, Mitiglinide), Alpha-glucosidase inhibitors (e.g. Acarbose,Miglitol, Voglibose), Amylin and Amylin analogues (e.g. Pramlintide,Symlin).

GPR119 agonists (e.g. GSK-263A, PSN-821, MBX-2982, APD-597, ZYG-19,DS-8500), GPR40 agonists (e.g. Fasiglifam/TAK-875, TUG-424, P-1736,JTT-851, GW9508).

Other suitable combination partners are: Cycloset, inhibitors of11-beta-HSD (e.g. LY2523199, BMS770767, RG-4929, BMS816336, AZD-8329,HSD-016, BI-135585), activators of glucokinase (e.g. TTP-399, AMG-151,TAK-329, GKM-001), inhibitors of DGAT (e.g. LCQ-908), inhibitors ofprotein tyrosinephosphatase 1 (e.g. Trodusquemine), inhibitors ofglucose-6-phosphatase, inhibitors of fructose-1,6-bisphosphatase,inhibitors of glycogen phosphorylase, inhibitors of phosphoenol pyruvatecarboxykinase, inhibitors of glycogen synthase kinase, inhibitors ofpyruvate dehydrokinase, alpha2-antagonists, CCR-2 antagonists, SGLT-1inhibitors (e.g. LX-2761), dual SGLT2/SGLT1 inhibitors.

One or more lipid lowering agents are also suitable as combinationpartners, such as for example: HMG-CoA-reductase inhibitors (e.g.Simvastatin, Atorvastatin), fibrates (e.g. Bezafibrate, Fenofibrate),nicotinic acid and the derivatives thereof (e.g. Niacin), PPAR-(alpha,gamma or alpha/gamma) agonists or modulators (e.g. Aleglitazar),PPAR-delta agonists, ACAT inhibitors (e.g. Avasimibe), cholesterolabsorption inhibitors (e.g. Ezetimibe), Bile acid-binding substances(e.g. Cholestyramine), ileal bile acid transport inhibitors, MTPinhibitors, or modulators of PCSK9.

HDL-raising compounds such as: CETP inhibitors (e.g. Torcetrapib,Anacetrapid, Dalcetrapid, Evacetrapid, JTT-302, DRL-17822, TA-8995) orABC1 regulators.

Other suitable combination partners are one or more active substancesfor the treatment of obesity, such as for example: Sibutramine,Tesofensine, Orlistat, antagonists of the cannabinoid-1 receptor, MCH-1receptor antagonists, MC4 receptor agonists, NPY5 or NPY2 antagonists(e.g. Velneperit), beta-3-agonists, leptin or leptin mimetics, agonistsof the 5HT2c receptor (e.g. Lorcaserin), or the combinations ofbupropione/naltrexone, bupropione/zonisamide, bupropione/phentermine orpramlintide/metreleptin.

Other suitable combination partners are:

Further gastrointestinal peptides such as Peptide YY 3-36 (PYY3-36) oranalogues thereof, pancreatic polypeptide (PP) or analogues thereof.

Glucagon receptor agonists or antagonists, GIP receptor agonists orantagonists, ghrelin antagonists or inverse agonists, Xenin andanalogues thereof.

Moreover, combinations with drugs for influencing high blood pressure,chronic heart failure or atherosclerosis, such as e.g. Angiotensin IIreceptor antagonists (e.g. telmisartan, candesartan, valsartan,losartan, eprosartan, irbesartan, olmesartan, tasosartan, azilsartan),ACE inhibitors, ECE inhibitors, diuretics, beta-blockers, calciumantagonists, centrally acting hypertensives, antagonists of thealpha-2-adrenergic receptor, inhibitors of neutral endopeptidase,thrombocyte aggregation inhibitors and others or combinations thereofare suitable.

In another aspect, this invention relates to the use of a compoundaccording to the invention or a physiologically acceptable salt thereofcombined with at least one of the active substances described above as acombination partner, for preparing a medicament which is suitable forthe treatment or prevention of diseases or conditions which can beaffected by binding to the receptors for GLP-1 and glucagon and bymodulating their activity. This is preferably a disease in the contextof the metabolic syndrome, particularly one of the diseases orconditions listed above, most particularly diabetes or obesity orcomplications thereof.

The use of the compounds according to the invention, or aphysiologically acceptable salt thereof, in combination with one or moreactive substances may take place simultaneously, separately orsequentially.

The use of the compound according to the invention, or a physiologicallyacceptable salt thereof, in combination with another active substancemay take place simultaneously or at staggered times, but particularlywithin a short space of time. If they are administered simultaneously,the two active substances are given to the patient together; if they areused at staggered times, the two active substances are given to thepatient within a period of less than or equal to 12 hours, butparticularly less than or equal to 6 hours.

Consequently, in another aspect, this invention relates to a medicamentwhich comprises a compound according to the invention or aphysiologically acceptable salt of such a compound and at least one ofthe active substances described above as combination partners,optionally together with one or more inert carriers and/or diluents.

The compound according to the invention, or physiologically acceptablesalt or solvate thereof, and the additional active substance to becombined therewith may both be present together in one formulation, forexample a in a vial or a cartridge, or separately in two identical ordifferent formulations, for example as so-called kit-of-parts.

LEGENDS TO THE FIGURES

FIG. 1. Body weight development during 4 weeks of subcutaneous treatmentwith SEQ ID NO: 7 and SEQ ID NO: 8, 50 μg/kg bid in female high-fat fedC57BL/6 mice. Data are mean+SEM.

FIG. 2. Relative body weight change in % during 4 weeks of subcutaneoustreatment with SEQ ID NO: 7 and SEQ ID NO: 8, 50 μg/kg bid in femalehigh-fat fed C57BL/6 mice. Data are mean+SEM.

FIG. 3. Determination of total fat mass measured by nuclear magneticresonance (NMR), two days before and after 4 weeks of treatment with SEQID NO: 7 and SEQ ID NO: 8, 50 μg/kg bid in female high-fat fed C57BL/6mice. Data are mean+SEM.

FIG. 4. Acute effect of s.c. administration of compound SEQ ID NO: 7 andSEQ ID NO: 8 50 μg/kg on blood glucose in female high-fat fed C57BL/6mice. Data are mean+SEM.

FIG. 5. Acute effect of s.c. administration of compound SEQ ID NO: 15,50 μg/kg qd on blood glucose in female high-fat fed C57BL/6 mice. Dataare mean+SEM.

FIG. 6. Relative body weight change in % during 4 weeks of subcutaneoustreatment with SEQ ID NO: 15, 50 μg/kg qd in female high-fat fed C57BL/6mice. Data are mean+SEM.

FIG. 7. Determination of total fat mass measured by nuclear magneticresonance (NMR), two days before and after 4 weeks of treatment with SEQID NO: 15, 50 μg/kg qd in female high-fat fed C57BL/6 mice. Data aremean+SEM.

METHODS Abbreviations Employed are as Follows

-   AA amino acid-   AEEAc (2-(2-aminoethoxy)ethoxy)acetyl-   cAMP cyclic adenosine monophosphate-   Boc tert-butyloxycarbonyl-   BOP (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium    hexafluorophosphate-   BSA bovine serum albumin-   tBu tertiary butyl-   DCM dichloromethane-   Dde 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl-   ivDde 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butyl-   DIC N,N′-diisopropylcarbodiimide-   DIPEA N,N-diisopropylethylamine-   DMEM Dulbecco's modified Eagle's medium-   DMF dimethyl formamide-   DMS dimethylsulfide-   EDT ethanedithiol-   FA formic acid-   FBS fetal bovine serum-   Fmoc fluorenylmethyloxycarbonyl-   HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HBSS Hanks' Balanced Salt Solution-   HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium    hexafluorophosphate-   HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid-   HOBt 1-hydroxybenzotriazole-   HOSu N-hydroxysuccinimide-   HPLC High Performance Liquid Chromatography-   HTRF Homogenous Time Resolved Fluorescence-   IBMX 3-isobutyl-1-methylxanthine-   LC/MS Liquid Chromatography/Mass Spectrometry-   Mint monomethoxy-trityl-   Palm palmitoyl-   PBS phosphate buffered saline-   PEG polyethylene glycole-   PK pharmacokinetic-   RP-HPLC reversed-phase high performance liquid chromatography-   Stea stearyl-   TFA trifluoro acetic acid-   Trt trityl-   UV ultraviolet-   γ-E γ-Glutamate

General Synthesis of Peptidic Compounds

Materials

Different Rink-Amide resins(4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethylresin, Merck Biosciences;4-[(2,4-Dimethoxyphenyl)(Fmoc-amino)methyl]phenoxy acetamido methylresin, Agilent Technologies) were used for the synthesis of peptideamides with loadings in the range of 0.2-0.7 mmol/g.

Fmoc protected natural amino acids were purchased from ProteinTechnologies Inc., Senn Chemicals, Merck Biosciences, Novabiochem, IrisBiotech, Bachem, Chem-Impex International or MATRIX Innovation. Thefollowing standard amino acids were used throughout the syntheses:Fmoc-L-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH,Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH,Fmoc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH,Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH,Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH,Fmoc-L-Trp(Boc)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OH.

In addition, the following special amino acids were purchased from thesame suppliers as above: Fmoc-L-Lys(ivDde)-OH, Fmoc-L-Lys(Mmt)-OH,Fmoc-Aib-OH, Fmoc-D-Ser(tBu)-OH, Fmoc-D-Ala-OH, Boc-L-His(Boc)-OH(available as toluene solvate) and Boc-L-His(Trt)-OH.

The solid phase peptide syntheses were performed for example on aPrelude Peptide Synthesizer (Protein Technologies Inc) or similarautomated synthesizer using standard Fmoc chemistry and HBTU/DIPEAactivation. DMF was used as the solvent. Deprotection: 20%piperidine/DMF for 2×2.5 min. Washes: 7×DMF Coupling 2:5:10 200 mMAA/500 mM HBTU/2M DIPEA in DMF 2× for 20 min. Washes: 5×DMF.

In cases where a Lys-side-chain was modified, Fmoc-L-Lys(ivDde)-OH orFmoc-L-Lys(Mmt)-OH was used in the corresponding position. Aftercompletion of the synthesis, the ivDde group was removed according to amodified literature procedure (S. R. Chhabra et al., Tetrahedron Lett.39, (1998), 1603), using 4% hydrazine hydrate in DMF. The Mmt group wasremoved by repeated treatment with 1% TFA in dichloromethane. Thefollowing acylations were carried out by treating the resin with theN-hydroxy succinimide esters of the desired acid or using couplingreagents like HBTU/DIPEA or HOBt/DIC.

All the peptides that have been synthesized were cleaved from the resinwith King's cleavage cocktail consisting of 82.5% TFA, 5% phenol, 5%water, 5% thioanisole, 2.5% EDT. The crude peptides were thenprecipitated in diethyl or diisopropyl ether, centrifuged, andlyophilized Peptides were analyzed by analytical HPLC and checked by ESImass spectrometry. Crude peptides were purified by a conventionalpreparative RP-HPLC purification procedure.

Alternatively, peptides were synthesized by a manual synthesisprocedure:

0.3 g Desiccated Rink amide MBHA Resin (0.66 mmol/g) was placed in apolyethylene vessel equipped with a polypropylene filter. Resin wasswelled in DCM (15 ml) for 1 h and DMF (15 ml) for 1 h. The Fmoc groupon the resin was de-protected by treating it twice with 20% (v/v)piperidine/DMF solution for 5 and 15 min. The resin was washed withDMF/DCM/DMF (6:6:6 time each). A Kaiser test (quantitative method) wasused for the conformation of removal of Fmoc from solid support. TheC-terminal Fmoc-amino acid (5 equiv. excess corresponding to resinloading) in dry DMF was added to the de-protected resin and coupling wasinitiated with 5 equivalent excess of DIC and HOBT in DMF. Theconcentration of each reactant in the reaction mixture was approximately0.4 M. The mixture was rotated on a rotor at room temperature for 2 h.Resin was filtered and washed with DMF/DCM/DMF (6:6:6 time each). Kaisertest on peptide resin aliquot upon completion of coupling was negative(no colour on the resin). After the first amino acid attachment, theunreacted amino group, if any, in the resin was capped used aceticanhydride/pyridine/DCM (1:8:8) for 20 minutes to avoid any deletion ofthe sequence. After capping, resin was washed with DCM/DMF/DCM/DMF(6/6/6/6 time each). The Fmoc group on the C-terminal amino acidattached peptidyl resin was deprotected by treating it twice with 20%(v/v) piperidine/DMF solution for 5 and 15 min. The resin was washedwith DMF/DCM/DMF (6:6:6 time each). The Kaiser test on peptide resinaliquot upon completion of Fmoc-deprotection was positive.

The remaining amino acids in target sequence on Rink amide MBHA Resinwere sequentially coupled using Fmoc AA/DIC/HOBt method using 5equivalent excess corresponding to resin loading in DMF. Theconcentration of each reactant in the reaction mixture was approximately0.4 M. The mixture was rotated on a rotor at room temperature for 2 h.Resin was filtered and washed with DMF/DCM/DMF (6:6:6 time each). Aftereach coupling step and Fmoc deprotection step, a Kaiser test was carriedout to confirm the completeness of the reaction.

After the completion of the linear sequence, the ε-amino group of lysineused as branching point or modification point was deprotected by using2.5% hydrazine hydrate in DMF for 15 min×2 and washed with DMF/DCM/DMF(6:6:6 time each). The γ-carboxyl end of glutamic acid was attached tothe ε-amino group of Lys using Fmoc-Glu(OH)-OtBu with DIC/HOBt method (5equivalent excess with respect to resin loading) in DMF. The mixture wasrotated on a rotor at room temperature for 2 h. The resin was filteredand washed with DMF/DCM/DMF (6×30 ml each). The Fmoc group on theglutamic acid was de-protected by treating it twice with 20% (v/v)piperidine/DMF solution for 5 and 15 min (25 ml each). The resin waswashed with DMF/DCM/DMF (6:6:6 time each). A Kaiser test on peptideresin aliquot upon completion of Fmoc-deprotection was positive.

If the side-chain branching also contains one more γ-glutamic acid, asecond Fmoc-Glu(OH)-OtBu used for the attachment to the free amino groupof γ-glutamic acid with DIC/HOBt method (5 equivalent excess withrespect to resin loading) in DMF. The mixture was rotated on a rotor atroom temperature for 2 h. Resin was filtered and washed with DMF/DCM/DMF(6×30 ml each). The Fmoc group on the γ-glutamic acid was de-protectedby treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15min (25 mL). The resin was washed with DMF/DCM/DMF (6:6:6 time each). AKaiser test on peptide resin aliquot upon completion ofFmoc-deprotection was positive.

Palmitic Acid & Stearic Acid Attachment to Side Chains of Glutamic Acid:

To the free amino group of γ-glutamic acid, palmitic acid or stearicacid (5 equiv.) dissolved in DMF was added and coupling was initiated bythe addition of DIC (5 equiv.) and HOBt (5 equiv.) in DMF. The resin waswashed with DMF/DCM/DMF (6:6:6 time each).

Final Cleavage of Peptide from the Resin:

The peptidyl resin synthesized by manual synthesis was washed with DCM(6×10 ml), MeOH (6×10 ml) and ether (6×10 ml) and dried in vacuumdesiccators overnight. The cleavage of the peptide from the solidsupport was achieved by treating the peptide-resin with reagent cocktail(80.0% TFA/5% thioanisole/5% phenol/2.5% EDT, 2.5% DMS and 5% DCM) atroom temperature for 3 h. Cleavage mixture was collected by filtrationand the resin was washed with TFA (2 ml) and DCM (2×5 ml). The excessTFA and DCM was concentrated to small volume under nitrogen and a smallamount of DCM (5-10 ml) was added to the residue and evaporated undernitrogen. The process was repeated 3-4 times to remove most of thevolatile impurities. The residue was cooled to 0° C. and anhydrous etherwas added to precipitate the peptide. The precipitated peptide wascentrifuged and the supernatant ether was removed and fresh ether wasadded to the peptide and re-centrifuged. The crude sample waspreparative HPLC purified and lyophilized. The identity of peptide wasconfirmed by LCMS.

Analytical HPLC/UPLC

Method A: Detection at 210-225 nm

-   column: Waters ACQUITY UPLC® CSH™ C18 1.7 μm (150×2.1 mm) at 50° C.-   solvent: H₂O+0.5% TFA:ACN+0.35% TFA (flow 0.5 ml/min)-   gradient: 80:20 (0 min) to 80:20 (3 min) to 25:75 (23 min) to 2:98    (23.5 min) to 2:98 (30.5 min) to 80:20 (31 min) to 80:20 (37 min)-   optionally with mass analyser: LCT Premier, electrospray positive    ion mode

Method B: Detection at 210-225 nm

-   column: Aries prep XBC 18 (4.6×250 mm×3.6 μm), Temp: 25° C.-   solvent: H₂O+0.1% TFA:ACN+0.1% TFA (flow 1 ml/min)-   gradient: Equilibration of the column with 2% buffer B and elution    by a gradient of 2% to 70% buffer B during 15 min.

General Preparative HPLC Purification Procedure

The crude peptides were purified either on an Äkta Purifier System, aJasco semiprep HPLC System or a Agilent 1100 HPLC system. PreparativeRP-C18-HPLC columns of different sizes and with different flow rateswere used depending on the amount of crude peptide to be purified.Acetonitrile+0.1% TFA (B) and water+0.1% TFA (A) were employed aseluents. Product-containing fractions were collected and lyophilized toobtain the purified product, typically as TFA salt.

Solubility and Stability-Testing of Exendin-4 Derivatives

Prior to the testing of solubility and stability of a peptide batch, itspurity (HPLC-UV) was determined.

For solubility testing, the target concentration was 10 mg purecompound/ml. Therefore, solutions from solid samples were prepared indifferent buffer systems with a concentration of 10 mg/mL compound basedon the previously determined % purity. HPLC-UV was performed after 2 hof gentle agitation from the supernatant, which was obtained by 20 minof centrifugation at 4500 rpm.

The solubility was then determined by comparison of a 0.2 μL-injectionwith the UV peak areas obtained with a stock solution of the peptide ata concentration of 1.2 mg/mL in DMSO (based on % purity), injectingvarious volumes ranging from 0.2-2 μl. This analysis also served asstarting point (t0) for the stability testing.

For stability testing, an aliquot of the supernatant obtained forsolubility was stored for 7 days at 40° C. After that time course, thesample was centrifuged for 20 min at 4500 rpm and 0.2 μL of thesupernatant were analysed with HPLC-UV.

For determination of the amount of the remaining peptide, the peak areasof the target compound at t0 and t7 were compared, resulting in “%remaining peptide”, following the equation% remaining peptide=[(peak area peptide t7)×100]/peak area peptide t0.

The stability is expressed as “% remaining peptide”.

As HPLC/UPLC method Method B has been used, detecting at 214 nM.

In Vitro Cellular Assays for GLP-1, Glucagon and GIP Receptor Efficacy

Agonism of compounds for the receptors was determined by functionalassays measuring cAMP response of HEK-293 cell lines stably expressinghuman GLP-1, GIP or glucagon receptor.

cAMP content of cells was determined using a kit from Cisbio Corp. (cat.no. 62AM4PEC) based on HTRF (Homogenous Time Resolved Fluorescence). Forpreparation, cells were split into T175 culture flasks and grownovernight to near confluency in medium (DMEM/10% FBS). Medium was thenremoved and cells washed with PBS lacking calcium and magnesium,followed by proteinase treatment with accutase (Sigma-Aldrich cat. no.A6964). Detached cells were washed and resuspended in assay buffer(1×HBSS; 20 mM HEPES, 0.1% BSA, 2 mM IBMX) and cellular densitydetermined. They were then diluted to 400000 cells/ml and 25 μl-aliquotsdispensed into the wells of 96-well plates. For measurement, 25 μl oftest compound in assay buffer was added to the wells, followed byincubation for 30 minutes at room temperature. After addition of HTRFreagents diluted in lysis buffer (kit components), the plates wereincubated for 1 hr, followed by measurement of the fluorescence ratio at665/620 nm. In vitro potency of agonists was quantified by determiningthe concentrations that caused 50% activation of maximal response(EC50).

Bioanalytical Screening Method for Quantification of Exendin-4Derivatives in Mice and Pigs

Mice were dosed 1 mg/kg subcutaneously (s.c.). The mice were sacrifiedand blood samples were collected after 0.25, 0.5, 1, 2, 4, 8, 16 and 24hours post application. Plasma samples were analyzed after proteinprecipitation via liquid chromatography mass spectrometry (LC/MS). PKparameters and half-life were calculated using WinonLin Version 5.2.1(non-compartment model).

Female Göttinger minipigs were dosed 0.1 mg/kg subcutaneously (s.c.).Blood samples were collected after 0.25, 0.5, 1, 2, 4, 8, 24, 32, 48, 56and 72 hours post application. Plasma samples were analyzed afterprotein precipitation via liquid chromatography mass spectrometry(LC/MS). PK parameters and half-life were calculated using WinonLinVersion 5.2.1 (non-compartment model).

Gastric Emptying and Intestinal Passage in Mice

Female NMRI-mice of a body weight between 20 and 30 g are used. Mice areadapted to housing conditions for at least one week.

Mice are overnight fasted, while water remains available all the time.On the study day, mice are weighed, single-caged and allowed access to500 mg of feed for 30 min, while water is removed. At the end of the 30min feeding period, remaining feed is removed and weighed. 60 min later,a coloured, non-caloric bolus is instilled via gavage into the stomach.The test compound/reference compound or its vehicle in the control groupis administered subcutaneously, to reach Cmax when coloured bolus isadministered. After another 30 min, the animals are sacrificed and thestomach and the small intestine prepared. The filled stomach is weighed,emptied, carefully cleaned and dried and reweighed. The calculatedstomach content indicates the degree of gastric emptying. The smallintestine is straightened without force and measured in length. Then thedistance from the gastric beginning of the gut to the tip of thefarthest travelled intestinal content bolus is measured. The intestinalpassage is given as relation in percent of the latter distance and thetotal length of the small intestine. Comparable data can be obtained forboth female and male mice.

Statistical analyses are performed with Everstat 6.0 by 1-way-ANOVA,followed by Dunnetts or Newman-Keuls as post-hoc test, respectively.Differences are considered statistically significant at the p<0.05level. As post hoc test Dunnet's Test is applied to compare versusvehicle control, only. Newman-Keul's Test is applied for all pairwisecomparisons (i.e. versus vehicle and reference groups).

Automated Assessment of Feed Intake in Mice

Female NMRI-mice of a body weight between 20 and 30 g are used. Mice areadapted to housing conditions for at least one week and for at least oneday single-caged in the assessment equipment, when basal data arerecorded simultaneously. On the study day, test product is administeredsubcutaneously close to the lights-off phase (12 h lights off) andassessment of feed consumption is directly started afterwards.Assessment included continued monitoring (every 30 min) over 22 hours.Repetition of this procedure over several days is possible. Restrictionof assessment to 22 hours is for practical reasons to allow forreweighing of animals, refilling of feed and water and drugadministration between procedures. Results can be assessed as cumulateddata over 22 hours or differentiated to 30 min intervals. Comparabledata can be obtained for both female and male mice.

Statistical analyses are performed with Everstat 6.0 by two-way ANOVA onrepeated measures and Dunnett's post-hoc analyses. Differences areconsidered statistically significant at the p<0.05 level.

Acute and Chronic Effects After Subcutaneous Treatment on Blood Glucoseand Body Weight in Female Diet-Induced Obese (DIO) C57BL/6 Mice

C57BL/6 Harlan mice are housed in groups in a specific pathogen-freebarrier facility on a 12 h light/dark cycle with free access to waterand standard or high-fat diet. After prefeeding on high-fat diet, miceare stratified to treatment groups (n=8), so that each group has similarmean body weight. An age-matched group with ad-libitum access tostandard chow is included as standard control group. Before theexperiment, mice are subcutaneously (s.c.) injected with vehiclesolution and weighed for 3 days to acclimate them to the procedures.

1) Acute effect on blood glucose in fed female DIO mice: initial bloodsamples are taken just before first administration (s.c.) of vehicle(phosphate buffer solution) or the exendin-4 derivatives (dissolved inphosphate buffer), respectively. The volume of administration is 5mL/kg. The animals have access to water and their corresponding dietduring the experiment. Blood glucose levels are measured at t=0 h, t=1h, t=2 h, t=3 h, t=4 h, t=6 h and t=24 h (method: Accu-Checkglucometer). Blood sampling is performed by tail incision withoutanaesthesia.

2) Chronic effect on body weight in female DIO mice: mice are treatedonce (s.c. in evening hours) or twice daily (s.c. in the morning and inthe evening), respectively, at the beginning and the end of the lightphase with either vehicle or exendin-4 derivatives for 4 weeks. Bodyweight is recorded daily. Two days before start of treatment and on day26, total fat mass is measured by nuclear magnetic resonance (NMR).

Statistical analyses are performed with Everstat 6.0 by repeatedmeasures two-way ANOVA and Dunnetts post-hoc analyses (glucose profile)and 1-way-ANOVA, followed by Dunnetts post-hoc test (body weight, bodyfat). Differences versus vehicle-treated DIO control mice are consideredstatistically significant at the p<0.05 level.

Effects of 4 Weeks of Treatment on Glucose, HbA1c and Oral GlucoseTolerance in Female Diabetic dbdb-Mice

8 week old, female diabetic dbdb-mice of mean non-fasted glucose valueof 14.5 mmol/l and a body weight of 37-40 g are used. Mice areindividually marked and are adapted to housing conditions for at leastone week.

7 days prior to study start, baseline values for non-fasted glucose andHbA1c are determined, 5 days prior to study start, mice are assigned togroups and cages (5 mice per cage, 10 per group) according to theirHbA1c values to ensure even distribution of lower and higher valuesbetween groups (stratification).

Mice are treated for 4 weeks, by twice daily subcutaneous administrationin the morning and the afternoon. Blood samples from the tail tip areobtained for HbA1c on study day 21 and oral glucose tolerance isassessed in the 4th week.

An oral glucose tolerance test is done in the morning without priorextra compound administration to majorly assess the effect of chronictreatment and less of acute compound administration. Mice are fasted for4 hours prior to oral glucose administration (2 g/kg, t=0 min). Bloodsamples are drawn prior to glucose administration and at 15, 30, 60, 90,120, and 180 min. Feed is returned after the last blood sampling.Results are represented as change from baseline, glucose in mmol/l andHbA1c in %.

Statistical analyses are performed with Everstat Version 6.0 based onSAS by 1-way-ANOVA, followed by Dunnett's post-hoc test againstvehicle-control. Differences are considered statistically significant atthe p<0.05 level.

Glucose Lowering in Non-Fasted Female Diabetic dbdb-Mice

Female diabetic dbdb-mice of mean non-fasted glucose value of 20-22mmol/l and a body weight of 42 g+/−0.6 g (SEM) are used. Mice areindividually marked and are adapted to housing conditions for at leastone week.

3-5 days prior to study start mice are assigned to groups and cages (4mice per cage, 8 per group) according to their non-fasted glucose valuesto ensure even distribution of lower and higher values between groups(stratification). On the study day, mice are weighed and dosed (t=0)Immediately prior to compound administration feed is removed while waterremains available, and a first blood sample at a tail incision is drawn(baseline). Further blood samples are drawn at the tail incision at 30,60, 90, 120, 240, 360, and 480 min.

Statistical analyses are performed with Everstat Version 6.0 based onSAS by 2-way-ANOVA on repeated measures, followed by Dunnett's post-hoctest against vehicle-control.

Differences are considered statistically significant at the p<0.05level.

EXAMPLES

The invention is further illustrated by the following examples.

Example 1 Synthesis of SEQ ID NO: 7

The manual synthesis procedure as described in Methods was carried outon a desiccated Rink amide MBHA Resin (0.66 mmol/g). The Fmoc-synthesisstrategy was applied with DIC/HOBt-activation. In position 14Fmoc-Lys(ivDde)-OH and in position 1 Boc-His(Boc)-OH were used. TheivDde-group was cleaved from the peptide on resin according to amodified literature procedure (S. R. Chhabra et al., Tetrahedron Lett.39, (1998), 1603), using 4% hydrazine hydrate in DMF. The peptide wascleaved from the resin with King's cocktail (D. S. King, C. G. Fields,G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crudeproduct was purified via preparative HPLC using an acetonitrile/watergradient (both buffers with 0.1% TFA). The purified peptide was analysedby LCMS (Method B). Deconvolution of the mass signals found under thepeak with retention time 14.29 min revealed the peptide mass 4649.20which is in line with the expected value of 4649.29.

Example 2 Synthesis of SEQ ID NO: 8

The manual synthesis procedure as described in Methods was carried outon a desiccated Rink amide MBHA Resin (0.66 mmol/g). The Fmoc-synthesisstrategy was applied with DIC/HOBT-activation. In position 14Fmoc-Lys(ivDde)-OH and in position 1 Boc-His(Boc)-OH were used. TheivDde-group was cleaved from the peptide on resin according to amodified literature procedure (S. R. Chhabra et al., Tetrahedron Lett.39, (1998), 1603), using 4% hydrazine hydrate in DMF. The peptide wascleaved from the resin with King's cocktail (D. S. King, C. G. Fields,G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crudeproduct was purified via preparative HPLC using an acetonitrile/watergradient (both buffers with 0.1% TFA). The purified peptide was analysedby LCMS (Method B). Deconvolution of the mass signals found under thepeak with retention time 14.05 min revealed the peptide mass 4634.80which is in line with the expected value of 4635.27.

In an analogous way, the peptides listed in Table 3 were synthesized andcharacterized.

TABLE 3 list of synthesized peptides and comparison of calculated vs.found molecular weight SEQ calc. found Monoisotopic or ID NO Mass massaverage mass 6 4545.4 4546.0 monoisotopic 7 4649.3 4649.2 average 84635.3 4634.8 average 9 4534.2 4533.0 average 10 4506.1 4504.8 average11 4520.2 4518.3 average 12 4662.4 4662.2 monoisotopic 13 4648.4 4648.4monoisotopic 14 4703.5 4703.6 monoisotopic 15 4689.5 4689.5 monoisotopic16 4793.5 4793.6 monoisotopic 17 4821.6 4821.6 monoisotopic 18 4837.64837.6 monoisotopic 20 4150.1 4150.2 monoisotopic 21 4675.5 4675.4monoisotopic 22 4689.5 4689.5 monoisotopic 23 4138.6 4139.4 average 244123.6 4122.6 average 25 4164.1 4164.1 monoisotopic

In an analogous way, the following peptides of Table 4 can besynthesized:

TABLE 4 List of peptides that can be synthesized in an analogous way.SEQ ID NO 19

Example 3 Stability and Solubility

Solubility and stability of peptidic compounds were assessed asdescribed in Methods. The results are given in Table 5.

TABLE 5 Stability and solubility SEQ Stability [%] solubility [mg/ml] IDNO pH 4.5 pH 7.4 pH 4.5 pH 7.4 7 89 100 >8 >8 8 98 100 >8 >8 14 93100 >8 >8 15 96 96 >8 >8

Example 4 In Vitro Data on GLP-1, Glucagon and GIP Receptor

Potencies of peptidic compounds at the GLP-1, glucagon and GIP receptorswere determined by exposing cells expressing human glucagon receptor(hGlucagon R), human GIP receptor (hGIP-R) or human GLP-1 receptor(hGLP-1 R) to the listed compounds at increasing concentrations andmeasuring the formed cAMP as described in Methods.

The results are shown in Table 6:

TABLE 6 EC50 values of exendin-4 derivatives at GLP-1, Glucagon and GIPreceptors (indicated in pM) SEQ ID EC50 EC50 EC50 NO hGLP-1R hGlucagon-RhGIP-R 1 0.4 >10000000 12500.0 6 14.1 136.0 2760.0 7 3.4 101.3 7617.5 82.3 28.1 2140.0 9 6.4 15.4 1160.0 10 3.5 52.4 890.0 11 10.3 261.013400.0 12 3.7 130.0 8290.0 13 1.8 22.8 2390.0 14 4.1 194.0 6530.0 152.4 42.6 1855.0 16 2.0 42.0 1870.0 17 2.8 16.2 906.0 18 8.3 8.6 2160.0

Example 5 Comparison Testing

A selection of inventive exendin-4 derivatives comprising afunctionalized amino acid in position 14 has been tested versuscorresponding compounds having in this position 14 a‘non-functionalized’ amino acid with otherwise identical amino acidsequence. The reference pair compounds and the corresponding EC50 valuesat GLP-1, Glucagon and GIP receptors (indicated in pM) are given inTable 7. As shown, the inventive exendin-4 derivatives show a superioractivity in comparison to the compounds with a ‘non-functionalized’amino acid in position 14.

Furthermore, a selection of inventive exendin-4 derivatives comprisingan Aib in position 27 has been tested versus corresponding compoundshaving in this position a lysine residue as in native exendin-4 andotherwise identical amino acid sequence. The reference pair compoundsand the corresponding EC50 values at GLP-1, Glucagon and GIP receptors(indicated in pM) are given in Table 8. As shown, the inventiveexendin-4 derivatives show a reduced activity on the GIP receptorcompared to the corresponding derivatives with Lys at position 27 as innative exendin-4.

TABLE 7 Comparison of exendin-4 derivatives comprising anon-functionalized amino acid in position 14 vs. exendin-4 derivativescomprising a functionalized amino acid in position 14 and otherwiseidentical amino acid sequence. EC50 values at GLP-1, Glucagon and GIPreceptors are indicated in pM. (K = lysine, Nle = norleucine, L =leucine, E-x53 = (S)-4-Carboxy-4-hexadecanoylamino-butyryl-, E-x70 =(S)-4-Carboxy-4-octadecanoylamino-butyryl-, Ac = acetate, E-E-x53 =(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-)SEQ ID EC50 EC50 EC50 residue in NO hGLP-1R hGlucagon-R hGIP-1 position14 24 8.0 3090.0 114000.0 L 23 52.5 42900.0 177000.0 K 8 2.3 28.1 2140.0K(γE-γE-x53) 9 6.4 15.4 1160.0 K(γE-x70) 10 3.5 52.4 890.0 K(γE-x53) 151.2 394.0 11900.0 L 21 1.2 3.2 193.0 K(γE-γE-x53)

TABLE 8 Comparison of exendin-4 derivatives comprising an Aib inposition 27 vs. exendin-4 derivatives comprising a Lys in position 27and otherwise identical amino acid sequence. EC50 values at GLP-1,Glucagon and GIP receptors are indicated in pM. SEQ ID EC50 EC50 EC50residue in NO hGLP-1R hGlucagon-R hGIP-1 position 27 21 1.2 3.2 193.0 K8 2.3 28.1 2140.0 Aib 22 1.8 5.9 148.0 K 7 3.4 101.3 7617.5 Aib

Example 6 Pharmacokinetic Testing

Pharmacokinetic profiles were determined as described in Methods.Calculated T_(1/2) and Cmax values are shown in Table 9.

TABLE 9 Pharmacokinetic profiles of exendin-4 derivatives. Mice (1mg/kg) Mini pigs (0.1 mg/kg) SEQ ID T_(1/2) Cmax T_(1/2) Cmax NO [h][ng/ml] [h] [ng/ml] 7 3.1 5510.0 29 667 8 3.8 4360.0 18.1 476

Example 7 Acute and Chronic Effects of SEQ ID NO: 7 and of SEQ ID NO: 8After Bid Subcutaneous Treatment on Blood Glucose and Body Weight inFemale Diet-Induced Obese (DIO) C57BL/6 Mice

1) Glucose Profile

After blood sampling to determine the blood glucose baseline level, feddiet-induced obese female C57BL/6 mice were administered 50 μg/kg of SEQID NO: 7, 50 μg/kg of SEQ ID NO: 8 or phosphate buffered solution(vehicle control on standard or high-fat diet) subcutaneously. Atpredefined time points, more blood samples were taken to measure bloodglucose and generate the blood glucose profile over 24 h.

SEQ ID NO: 7 and SEQ ID NO: 8 demonstrated a significant decrease inblood glucose compared to DIO control mice for time points t=1, 2, 3, 4,6 and 24 h post compound dosing (p<0.0001, 2-W-ANOVA-RM, post hocDunnett's Test; mean±SEM) (see FIG. 4).

2) Body Weight

Female obese C57BL/6 mice were treated for 4 weeks twice dailysubcutaneously with 50 μg/kg SEQ ID NO: 7, 50 μg/kg SEQ ID NO: 8 orvehicle. Body weight was recorded daily, and body fat content wasdetermined before the start and after 4 weeks of treatment. Treatmentwith 50 μg/kg SEQ ID NO: 7 showed a statistically significant decreasewhen compared to vehicle DIO control mice in daily body weight beginningon day 7 and continuing through the end of the study (p>0.0001 by theend of the study). Treatment with 50 μg/kg SEQ ID NO: 8 reduced bodyweight significantly when compared to vehicle DIO control mice beginningon day 5 and continuing through the end of the study (p>0.0001 by theend of the study, Table 10, FIGS. 1 and 2). These changes resulted froma decrease in body fat, as shown by the absolute changes in body fatcontent (Table 10, FIG. 3).

TABLE 10 Weight change in DIO mice over a 4-week treatment period (mean± SEM) Overall weight Body fat Example (Dose) change (g) change (g)Control standard diet +0.94 ± 0.4 +2.56 ± 0.4 Control high-fat diet+3.83 ± 0.5 +5.00 ± 0.5 SEQ ID NO: 7 (50 μg/kg bid) −5.49 ± 0.9 −3.73 ±0.8 SEQ ID NO: 8 (50 μg/kg bid) −5.38 ± 0.5 −3.81 ± 0.6

Example 8 Acute and Chronic Effects of SEQ ID NO: 15 After qdSubcutaneous Treatment on Blood Glucose and Body Weight in FemaleDiet-Induced Obese (DIO) C57BL/6 Mice

1) Glucose Profile

After blood sampling to determine the blood glucose baseline level, feddiet-induced obese female C57BL/6 mice were administered 50 μg/kg of SEQID NO: 15 or phosphate buffered solution (vehicle control on standard orhigh-fat diet) subcutaneously once daily. At predefined time points,more blood samples were taken to measure blood glucose and generate theblood glucose profile over 24 h.

SEQ ID NO: 15 demonstrated a significant decrease in blood glucosecompared to DIO control mice for time points t=1, 2, 3, 4, 6 and 24 hpost compound dosing (p<0.001, 2-W-ANOVA-RM, post hoc Dunnett's Test;mean±SEM) (see FIG. 5).

2) Body Weight

Female obese C57BL/6 mice were treated for 4 weeks once dailysubcutaneously with 50 μg/kg SEQ ID NO: 15 or vehicle. Body weight wasrecorded daily, and body fat content was determined before the start andafter 4 weeks of treatment.

Treatment with 50 μg/kg SEQ ID NO: 15 showed a statistically significantdecrease when compared to vehicle DIO control mice in daily body weightbeginning on day 4 and continuing through the end of the study (p>0.001by the end of the study, Table 11, FIG. 6). These changes resulted froma decrease in body fat, as shown by the absolute changes in body fatcontent (Table 11, FIG. 7).

TABLE 11 Weight change in DIO mice over a 4-week treatment period (mean± SEM) Example Overall weight Body fat (Dose) change (g) change (g)Control standard diet +1.71 ± 0.3 +0.71 ± 0.4  Control high-fat diet+5.41 ± 0.5 +3.26 ± 0.4  SEQ ID NO: 15 −4.59 ± 1.1 −4.01 ± 0.68 (50μg/kg qd)

TABLE 12 Sequences SEQ. ID sequence 1H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W-L-K-N-G-G-P-S-S-G-A-P-P-P- S-NH2 2H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A- K-E-F-I-A-W-L-V-K-G-R-NH2 3H-S-Q-G-T-F-T-S-D-Y-S-K-Y-L-D-S-R-R-A- Q-D-F-V-Q-W-L-M-N-T-OH 4H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K(γE-x53)-E-F-I-A-W-L-V-R-G-R-G-OH 5Y-A-E-G-T-F-I-S-D-Y-S-I-A-M-D-K-I-H-Q-Q-D-F-V-N-W-L-L-A-Q-K-G-K-K-N-D-W-K-H- N-I-T-Q-OH 6H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-x70)-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-A-dAla-G- P-S-S-G-A-P-P-P-S-NH2 7H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE- x53)-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-A-dAla-G-P-S-S-G-A-P-P-P-S-NH2 8 H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-A-G- G-P-S-S-G-A-P-P-P-S-NH2 9H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-x70)-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-A-G-G-P-S- S-G-A-P-P-P-S-NH2 10H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-x53)-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-A-G-G-P-S- S-G-A-P-P-P-S-NH2 11H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-x53)-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-A-dAla-G- P-S-S-G-A-P-P-P-S-NH2 12H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE- x53)-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-S-dAla-G-P-S-S-G-A-P-P-P-S-NH2 13 H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-S- G-G-P-S-S-G-A-P-P-P-S-NH2 14H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE- x53)-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-K-dAla-G-P-S-S-G-A-P-P-P-S-NH2 15 H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-K- G-G-P-S-S-G-A-P-P-P-S-NH2 16H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(AEEAc-AEEAc- E-x53)-D-E-Q-R-A-K-L-F-I-E-W- L-Aib-A-G-G-P-S-S-G-A-P-P-P-S-NH217 H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(AEEAc-AEEAc- E-x70)-D-E-Q-R-A-K-L-F-I-E-W- L-Aib-A-G-G-P-S-S-G-A-P-P-P-S-NH218 H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(AEEAc-AEEAc-AEEAc-x70)-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P-S-NH2 19H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(AEEAc-AEEAc- E-x99)-D-E-Q-R-A-K-L-F-I-E-W- L-Aib-A-G-G-P-S-S-G-A-P-P-P-S-NH220 H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-A-dAla-G-P-S- S-G-A-P-P-P-S-NH2 21H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E-Q-R-A-K-L-F-I-E-W-L-K-A-G-G- P-S-S-G-A-P-P-P-S-NH2 22H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E-Q-R-A-K-L-F-I-E-W-L-K-A-dAla- G-P-S-S-G-A-P-P-P-S-NH2 23H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P- P-P-S-NH2 24H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-L-D-E-Q-R-A-K-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P- P-P-S-NH2 25H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-L-D-E-Q-R-A-K-L-F-I-E-W-L-K-A-G-G-P-S-S-G-A-P-P- P-S-NH2

The invention claimed is:
 1. A peptidic compound of formula (I):

or a salt or solvate thereof, wherein: X14 is an amino acid residue witha functionalized —NH₂ side chain group selected from the groupconsisting of Lys, Orn, Dab, and Dap, wherein the —NH₂ side chain groupis functionalized by —Z—C(O)—R⁵, wherein Z is a linker comprising 1-5amino acid linker groups selected from the group consisting ofγ-glutamate (γE) and AEEAc and combinations thereof in allstereoisomeric forms, R⁵ is a moiety comprising up to 50 carbon atomsand heteroatoms selected from the group consisting of N and O, X28 is anamino acid residue selected from the group consisting of Ala, Lys, andSer, X29 is an amino acid residue selected from the group consisting ofD-Ala and Gly, and R¹ is NH₂ or OH.
 2. The compound or salt or solvatethereof of claim 1, wherein R¹ is NH₂.
 3. The compound or salt orsolvate thereof according to claim 1, wherein the peptidic compound hasa relative activity of at least 0.09% compared to that of naturalglucagon at the glucagon receptor.
 4. The compound or salt or solvatethereof according to claim 1, wherein the peptidic compound exhibits arelative activity of at least 0.1% compared to that of a glucagon-likepeptide (GLP-1)(7-36)-amide at the GLP-1 receptor.
 5. The compound orsalt or solvate thereof according to claim 1, wherein X14 is Lys,wherein the —NH₂ side chain group is functionalized with a group—Z—C(O)R⁵, wherein Z is a group selected from the group consisting ofγE, γE-γE, AEEAc-AEEAc-γE, and AEEAc-AEEAc-AEEAc, and R⁵ is a groupselected from the group consisting of pentadecanyl, heptadecanyl, and16-carboxy hexadecanyl.
 6. The compound or salt or solvate thereofaccording to claim 1, wherein X14 is Lys, wherein the —NH₂ side chaingroup is functionalized with a group —Z—C(O)R⁵, wherein Z is a groupselected from the group consisting of γE, γE-γE, AEEAc-AEEAc-γE, andAEEAc-AEEAc-AEEAc, and R⁵ is a group selected from the group consistingof pentadecanyl and heptadecanyl.
 7. The compound or salt or solvatethereof according to claim 1, wherein X14 is Lys, wherein the —NH₂ sidechain group is functionalized by a group selected from the groupconsisting of (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,(S)-4-Carboxy-4-octadecanoylamino-butyryl-,(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,and(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,[2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-,X28 is Ala, X29 is an amino acid residue selected from the groupconsisting of D-Ala and Gly, and R¹ is NH₂.
 8. The compound or salt orsolvate thereof according to claim 1, wherein X14 is Lys, wherein the—NH₂ side chain group is functionalized by(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,X28 is Ser, X29 is an amino acid residue selected from the groupconsisting of D-Ala and Gly, and R¹ is NH₂.
 9. The compound or salt orsolvate thereof according to claim 1, wherein X14 is Lys, wherein the—NH₂ side chain group is functionalized by(S)-4-carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,X28 is Lys, X29 is an amino acid residue selected from the groupconsisting of D-Ala and Gly, and R¹ is NH₂.
 10. The compound or salt orsolvate thereof according to claim 1, wherein X14 is Lys, wherein the—NH₂ side chain group is functionalized by a group selected from thegroup consisting of (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,(S)-4-Carboxy-4-octadecanoylamino-butyryl-, and(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,X28 is an amino acid residue selected from the group consisting of Ala,Lys, and Ser, X29 is D-Ala, and R¹ is NH₂.
 11. The compound or salt orsolvate thereof of claim 1, wherein X14 is Lys, wherein the —NH₂ sidechain group is functionalized by a group selected from the groupconsisting of (S)-4-Carboxy-4-hexadecanoylamino-butyryl-,(S)-4-Carboxy-4-octadecanoylamino-butyryl-,(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,and(2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl,[2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-,X28 is an amino acid residue selected from the group consisting of Ala,Lys, and Ser, X29 is Gly, and R¹ is NH₂.
 12. The compound or salt orsolvate thereof of claim 1, wherein X14 is Lys, wherein the —NH₂ sidechain group is functionalized by(S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-,X28 is Ala, X29 is an amino acid residue selected from the groupconsisting of Gly and D-Ala, and R¹ is NH₂.
 13. The compound or salt orsolvate thereof of claim 1, selected from the compounds of SEQ ID NOs:6-19, or a salt or solvate thereof.
 14. The compound or salt or solvatethereof of claim 13, selected from the compounds of SEQ ID NOs: 6-18, ora salt or solvate thereof.
 15. The compound, salt or solvate accordingto claim 1, wherein the compound is the amino acid sequence of SEQ IDNO:
 7. 16. The compound, salt or solvate according to claim 1, whereinthe compound is the amino acid sequence of SEQ ID NO:
 8. 17. A solvateof a compound of claim
 1. 18. A hydrate of a compound of claim
 1. 19. Apharmaceutical composition comprising one or more compounds of claim 1,or a salt or solvate thereof as an active ingredient and at least onepharmaceutically acceptable carrier.
 20. The pharmaceutical compositionaccording to claim 19, further comprising at least one additionaltherapeutically active agent, wherein the additional therapeuticallyactive agent is selected from the group consisting of: insulin andinsulin derivatives selected from the group consisting of insulinglargine, insulin glusiline, insulin detemir, insulin lispro, insulindegludec, insulin aspart, basal insulin and analogues thereof, pegylatedinsulin, recombinant human insulin, polysialated insulins, long-actinginsulin, NN1045, insulin in combination with pramlintide, PE0139,fast-acting and short-acting insulins, insulin hydrogel, oral insulin,inhalable insulin, transdermal insulin and sublingual insulin, andinsulin derivatives which are bonded to albumin or another protein by abifunctional linker; GLP-1; GLP-1 analogues; GLP-1 receptor agonistsselected from the group consisting of lixisenatide, exenatide, ITCA 650,AC-2993, liraglutide, semaglutide, taspoglutide, albiglutide,dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, Langlenatide,HM-11260C, CM-3, ORMD-0901, NN-9924, NN-9926, NN-9927, CVX-096, ZYOG-1,ZYD-1, GSK-2374697, DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022, TT-401,BHM-034, MOD-6030, CAM-2036, DA-15864, ARI-2651, ARI-2255, xtenylatedexenatide, xtenylated glucagon, and polymer bound derivatives thereof;dual GLP-1/GIP receptor agonists; dual GLP-1/glucagon receptor agonists;protein YY₃₋₃₆ (PYY3-36); pancreatic polypeptide; glucagon receptoragonists; GIP receptor agonists or antagonists; ghrelin antagonists orinverse agonists; xenin; dipeptidyl peptidase IV (DPP-IV) inhibitors;sodium glucose cotransporter 2 (SGLT2) inhibitors; dual SGLT2/SGLT1inhibitors; biguanides; thiazolidinediones; dual peroxisomeproliferator-activated receptor (PPAR) agonists; sulfonylureas;meglitinides; alpha-glucosidase inhibitors; amylin and pramlintide; Gprotein-coupled receptor 119 (GPR119) agonists; GPR40 agonists; GPR120agonists; GPR142 agonists; systemic or low-absorbable transmembrane Gprotein-coupled receptor 5 (TGR5) agonists; bromocriptine mesylate;inhibitors of 11-beta-hydroxysteroid dehydrogenase (HSD); activators ofglucokinase; inhibitors of diacylglycerol acyltransferase (DGAT);inhibitors of protein tyrosinephosphatase 1; inhibitors ofglucose-6-phosphatase; inhibitors of fructose-1,6-bisphosphatase;inhibitors of glycogen phosphorylase; inhibitors of phosphoenol pyruvatecarboxykinase; inhibitors of glycogen synthase kinase; inhibitors ofpyruvate dehydrogenase kinase; alpha2-antagonists; C—C motif receptor(CCR-2) antagonists; modulators of glucose transporter-4; somatostatinreceptor 3 agonists; 3-hydroxy-3-methyl-glutaryl-coenzyme A(HMG-CoA)-reductase inhibitors; fibrates; nicotinic acid and derivativesthereof; nicotinic acid receptor 1 agonists; PPAR-alpha, gamma, oralpha/gamma agonists or modulators; PPAR-delta agonists; acyl-CoAcholesterol acyltransferase (ACAT) inhibitors; cholesterol absorptioninhibitors; bile acid-binding substances; ileal bile acid transporter(IBAT) inhibitors; microsomal triglyceride transfer protein (MTP)inhibitors; modulators of proprotein convertase subtilisin/kinexin type9 (PCSK9); low-density lipoprotein (LDL) receptor up-regulators by liverselective thyroid hormone receptor 13 agonists; high-density lipoprotein(HDL)-raising compounds; lipid metabolism modulators; phospholipase A2(PLA2) inhibitors; apolipoprotein A1 (ApoA-1) enhancers; thyroid hormonereceptor agonists; cholesterol synthesis inhibitors; omega-3 fatty acidsand derivatives thereof; substances for the treatment of obesityselected from the group consisting of sibutramine, tesofensine,tetrahydrolipstatin, cannabinoid-1 (CB-1) receptor antagonists,melanin-concentrating hormone-1 (MCH-1) antagonists, melanocortin 4(MC4) receptor agonists and partial agonists, neuropeptide Y5 (NPY5) orNPY2 antagonists, NPY4 agonists, beta-3-agonists, leptin or leptinmimetics, agonists of the 5-hydroxy tryptophan 2c (5HT2c) receptor,combinations of bupropione/naltrexone, combinations ofbupropione/zonisamide, combinations of bupropione/phentermine,combinations of pramlintide/metreleptin, and combinations ofphentermine/topiramate; and lipase inhibitors; angiogenesis inhibitors;H3 antagonists; Agouti-related protein (AgRP) inhibitors; triplemonoamine uptake inhibitors; methionine aminopeptidase type 2 (MetAP2)inhibitors; nasal formulation of the calcium channel blocker diltiazem;antisense molecules against production of fibroblast growth factorreceptor 4; prohibitin targeting peptide-1; and drugs for influencinghigh blood pressure, chronic heart failure, or atherosclerosis selectedfrom the group consisting of angiotensin II receptor antagonists,angiotensin-converting-enzyme (ACE) inhibitors,endothelin-converting-enzyme (ECE) inhibitors, diuretics, beta-blockers,calcium antagonists, centrally acting hypertensives, antagonists of thealpha-2-adrenergic receptor, inhibitors of neutral endopeptidase, andthrombocyte aggregation inhibitors.
 21. A pharmaceutical compositioncomprising one or more compounds of claim 1, or a physiologicallyacceptable salt or hydrate thereof as an active ingredient and at leastone pharmaceutically acceptable carrier.
 22. A method for the treatmentof hyperglycemia, type 1 diabetes, type 2 diabetes, or obesity, whichcomprises administering to a patient in need of such treatment aneffective amount of one or more compounds, salts or solvates of claim 1.23. The method of claim 22, for the treatment or prevention ofhyperglycemia, or type 2 diabetes.
 24. A method for the simultaneoustreatment of diabetes and obesity which comprises administering to apatient in need of such treatment an effective amount of one or morecompounds, salts or solvates of claim
 1. 25. A method for the treatmenthyperglycemia, type 1 diabetes, type 2 diabetes, or obesity, whichcomprises administering to a patient in need of such treatment aneffective amount of a pharmaceutical composition of claim
 19. 26. Themethod of claim 25 for the treatment of hyperglycemia or type 2diabetes.
 27. A method for the simultaneous treatment of diabetes andobesity which comprises administering to a patient in need of suchtreatment an effective amount of a pharmaceutical composition of claim19.